Resource utilization method of aminoglycoside antibiotic fermentation solid residues

Abstract

The invention discloses a resource utilization method of aminoglycoside antibiotic fermentation solid residues, which comprises the following steps: carrying out solid-liquid separation on adsorption fermentation waste liquid obtained by adsorption of aminoglycoside antibiotic fermentation liquid by resin to obtain fermentation solid residues, mixing the fermentation solid residues with soybean cake powder, sieving, and drying to obtain a self-made organic nitrogen source; and preparing a fermentation basic culture medium instead of soybean cake powder for fermentation to produce aminoglycoside antibiotics. On the premise that the fermentation result is not affected, the use amount and production cost of the soybean cake powder are saved, meanwhile, the discharge amount and treatment difficulty of the fermentation solid residues are reduced, and resource utilization of the antibiotic fermentation solid residues is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of antibiotic production, and particularly relates to a method for resource utilization of fermentation solid residues of aminoglycoside antibiotics. Background Art

[0002] In China, the production and demand for antibiotics are both large. During the fermentation production process, a large amount of bacterial residues will be generated. Since there are corresponding residual antibiotics in the bacterial residues, they are hazardous solid wastes. Once not properly treated, serious environmental problems will be caused. The treatment of a large amount of antibiotic bacterial residues has become a major problem.

[0003] Currently, the traditional treatment methods for solid hazardous wastes are incineration or landfill. Antibiotic bacterial residues were once used as organic fertilizers or feed additives, but now they belong to toxic solid wastes. When landfilling the bacterial residues, the residual antibiotics will not only cause the generation of environmental drug resistance, but may also accumulate in animals and plants through the soil and be transferred to the human body through the food chain, endangering human health; the use of antibiotic bacterial residues in feed and animal drinking water has been prohibited since 2002. The incineration method not only has huge costs, but also has an adverse impact on the atmosphere.

[0004] Antibiotic bacterial residues contain rich microbial proteins, residual culture media, antibiotics and other metabolites, and also contain trace elements such as Ca, P and S. Therefore, the common reutilization methods of current antibiotic bacterial residues are used as culture medium units, anaerobic fermentation and pyrolysis for energy utilization; used as feed additives, biosorbents, etc.

[0005] Cai Xiang et al. studied the use of kirromycin bacterial residues to replace the organic nitrogen source in the fermentation medium. When the bacterial residues were used as the sole nitrogen source, the addition amount of 4% had the highest titer, and the production cost was reduced, solving the problem of bacterial residue treatment. However, the antibiotic yield decreased slightly (Cai Xiang, Hao Youyu, Liu Xinxing, et al. Research on the reuse of kirromycin bacterial residues as nitrogen sources [J]. Chinese Journal of Antibiotics, 2011, 36(6): 478 - 481.).

[0006] Chinese Patent CN116042752A discloses a method for recycling apramycin fermentation waste liquid. The fermentation waste liquid separated after the first fermentation of apramycin is put into a fermentation tank and various raw materials are added to make a secondary fermentation medium, and then the production strain is inoculated again for secondary fermentation. Without affecting the fermentation level, the discharge of fermentation waste liquid and bacterial solid residues is significantly reduced, and the treatment pressure of fermentation wastewater is reduced.

[0007] However, at present, a large amount of flocculant needs to be added during the solid-liquid separation of antibiotic bacterial residues. However, when the addition amount of the flocculant is too large, it has a great inhibitory and toxic effect on the growth of microorganisms, seriously affecting the growth of the bacterial cells. However, without adding the flocculant, effective solid-liquid separation cannot be achieved. In addition, when directly using the bacterial residues as a nitrogen source to prepare a fermentation medium, the medium is relatively viscous, which will have a great impact on the properties of the fluid and oxygen supply, thereby affecting the growth of the bacterial cells and ultimately reducing the fermentation titer. Therefore, it is necessary to treat the antibiotic bacterial residues to reduce their impact on fermentation during the process of resource utilization. Summary of the Invention

[0008] In view of the above technical problems, the present invention provides a method for resource utilization of fermentation solid residues of aminoglycoside antibiotics. The fermentation solid residues obtained by solid-liquid separation are mixed with soybean cake powder and dried to prepare a self-made organic nitrogen source, and then antibiotic fermentation is carried out. Without affecting the fermentation results, the dosage of soybean cake powder and production cost are saved, and at the same time, the discharge amount and treatment difficulty of the fermentation solid residues are reduced, realizing the resource utilization of the fermentation solid residues of antibiotics.

[0009] In order to achieve the above object, the present invention provides a method for resource utilization of fermentation solid residues of aminoglycoside antibiotics, including the following steps: (1) Collecting fermentation solid residues: The adsorption fermentation waste liquid of aminoglycoside antibiotics is subjected to solid-liquid separation to obtain fermentation solid residues; (2) Mixing, sieving and drying the fermentation solid residues with soybean cake powder to obtain a self-made organic nitrogen source; (3) Replacing soybean cake powder with the self-made organic nitrogen source to prepare a fermentation basal medium; (4) Inoculating a seed liquid into the fermentation basal medium, fermenting and extracting to obtain aminoglycoside antibiotics.

[0010] Preferably, the aminoglycoside antibiotic in step (1) is any one of apramycin, gentamicin and neomycin.

[0011] Preferably, the solid-liquid separation method in step (1) is scraper centrifugation and disc centrifugation.

[0012] Further preferably, a flocculant can be added to the fermentation adsorption waste liquid before solid-liquid separation, and the flocculant is PAM.

[0013] Even more preferably, the flocculant is prepared into a mother liquor for use, and the concentration of the mother liquor is 1‰.

[0014] Even more preferably, the volume ratio of the fermentation adsorption waste liquid to the flocculant mother liquor is 16:1.

[0015] Preferably, the water content of the fermentation solid residues in step (1) is 40-50%.

[0016] Preferably, the mass ratio of the fermented solid residue to the soybean cake powder in step (2) is 1:1-5.

[0017] Preferably, the sieve mesh during sieving in step (2) is 20 mesh, and the drying conditions are drying at 40-50 °C for 12-20 h, and turning over every 3 h during drying.

[0018] Preferably, the water content of the self-made organic nitrogen source in step (2) is 5-10%.

[0019] Preferably, when the self-made organic nitrogen source replaces the soybean cake powder in step (3), the dosage is 90-100% of the dosage of the soybean cake powder.

[0020] Preferably, the volume ratio of the seed liquid to the fermentation basal medium in step (4) is 1:8-10.

[0021] Preferably, the fermentation conditions in step (4) are a tank temperature of 36±0.5 °C, an air volume of 0-40 L / min, and a rotation speed of 200-500 rpm.

[0022] The beneficial effects of the present invention are as follows: 1. Without using flocculants, only physical methods are used for the solid-liquid separation of the fermented waste liquid, avoiding the toxic effect of flocculants on microorganisms, thus avoiding the impact on fermentation, and at the same time saving production costs.

[0023] 2. Mixing and drying the fermented solid residue with the soybean cake powder to prepare a self-made organic nitrogen source, saving the dosage of the nitrogen source, reducing the discharge amount and treatment difficulty of the fermented solid residue; compared with the direct utilization of the fermented solid residue or the adsorbed fermented waste liquid, the contamination rate is reduced, thus reducing the requirements for the storage environment. In addition, the soybean cake powder in the self-made organic nitrogen source adsorbs the viscous substances in the fermented solid residue, reducing the caking rate and making the particle size more uniform, which is convenient for subsequent mixing with other raw materials.

[0024] 3. The present invention realizes the resource utilization of the fermented solid residue of aminoglycoside antibiotics. On the premise that the product quality is qualified, the yield is improved to a certain extent, and the method is simple and has a wide application range. Specific Embodiments

[0025] The technical solutions of the present invention will be further explained and illustrated below with specific embodiments. It should be noted that the following embodiments are only the preferred embodiments of the present invention and should not be construed as limiting the present invention. The protection scope of the present invention should be based on the content recorded in the claims. Modifications and substitutions made by those skilled in the art to the technical solutions of the present invention without creative work fall within the protection scope of the present invention.

[0026] Example 1 Taking gentamicin as an example, the steps to obtain the fermentation residue are as follows: (1) Inoculate the sandy soil spores onto the slant. After 12 days of cultivation, slant spores are obtained. Pour sterilized water onto the slant and dig the medium spores to make a spore suspension; (2) Inoculate the slant spore suspension into a 15L seed tank filled with the seed medium. Control the tank temperature at 35 ± 0.5°C, adjust the air volume to be controlled within 0 - 40 L / min, and the rotation speed at 200 - 500 rpm for stable cultivation for 32 - 34 h to obtain the seed liquid; (3) Transfer 5L of the seed liquid from the seed tank into a 50L fermentation tank filled with the fermentation basal medium. Control the tank temperature at 35 ± 0.5°C, adjust the air volume to be controlled within 0 - 40 L / min, and the rotation speed at 300 rpm for stable fermentation; During the fermentation process, supplement the basal material according to the metabolic situation; The formula of the fermentation basal medium consists of the following raw materials by weight percentage: 3.5% soybean cake powder, 5.5% starch, 1.8% glucose, 0.4% fish meal, 0.3% peptone, 0.05% ammonium sulfate, 0.05% potassium nitrate, 0.7% calcium carbonate, 0.8 mg cobalt chloride, 0.3% silicone oil, and the pH value is 7.0 - 7.2; (4) Stop fermentation when it reaches about 150 h. Take the fermentation broth from the fermentation tank, add ammonium-type 732 resin to adsorb gentamicin, and then use a 40-mesh sieve to filter and wash to obtain the resin and the adsorbed fermentation waste liquid respectively; (5) Send the adsorbed fermentation waste liquid into a scraper centrifuge to remove large particle solids such as broken resin and dry base skin, and then separate it by a disc centrifuge to obtain the fermentation solid residue with a water content of 50%.

[0027] Example 2 Use the fermentation solid residue prepared in Example 1 to prepare a self-made organic nitrogen source. The specific steps are as follows: (1) Mix the fermentation solid residue with soybean cake powder with a weight twice that of the fermentation solid residue, stir evenly, and then pass through a 20-mesh sieve to obtain the wet powder of the organic nitrogen source; (2) Dry the wet powder of the organic nitrogen source at 45°C for 15 h, and turn it over every 3 h during the drying process to prepare a self-made organic nitrogen source with a water content of 7%.

[0028] Example 3 The method and steps are the same as those in Example 2, only change the weight ratio of the fermentation solid residue to soybean cake powder to 1:1.5, and prepare a self-made organic nitrogen source with a water content of 7%.

[0029] Example 4 The method and steps are the same as those in Example 2, only change the weight ratio of the fermentation solid residue to soybean cake powder to 1:5, and prepare a self-made organic nitrogen source with a water content of 7%.

[0030] Example 5 The method and steps are the same as those in Example 2, except that the water content of the self-made organic nitrogen source is controlled at 10%.

[0031] Example 6 The method and steps are the same as those in Example 2, except that the water content of the self-made organic nitrogen source is controlled at 5%.

[0032] Comparative Example 1 The method and steps are the same as those in Example 2, except that the self-made organic nitrogen source is prepared by drying the fermented solid residue.

[0033] Comparative Example 2 The method and steps are the same as those in Example 2, except that the weight ratio of the fermented solid residue to the soybean cake powder is changed to 1:7 to prepare the self-made organic nitrogen source.

[0034] Comparative Example 3 The method and steps are the same as those in Example 2, except that the fermented solid residue is directly used as the self-made organic nitrogen source without operations such as drying.

[0035] Comparative Example 4 The method and steps are the same as those in Example 2, except that the water content of the self-made organic nitrogen source is controlled at 2%.

[0036] Comparative Example 5 The method and steps are the same as those in Example 2, except that the water content of the self-made organic nitrogen source is controlled at 12%.

[0037] Result detection: Take the self-made organic nitrogen sources prepared in Examples 2 - 6 and Comparative Examples 1 - 5, and conduct statistics on the water content, total nitrogen, fineness, and the situation of bacterial contamination after sterilization. Among them, the method for the bacterial contamination verification experiment is to take 20 g of the self-made organic nitrogen source and add 100 mL of water to prepare a suspension, sterilize it at 121 °C for 30 min, take 1 mL of the suspension in a laminar flow hood and add it to 10 mL of a sterile broth medium (beef extract 0.5%, peptone 0.5%, glucose 1%, sodium chloride 0.5%, phenol red 1%, adjust the pH to 6.5 - 6.8), and conduct disinfection and sterility inspection on the sterile broth; after the sterile broth is prepared, sterilize it with steam at 121.0 ± 1.0 °C and 0.100 ± 0.010 MPa for 30 minutes, place it in a constant temperature room at 37 °C for two days, and it can be used only after passing the bacterial inspection. The sterile broth after adding the suspension sample is cultured at 37 °C, and microscopic examination is carried out after 24 h.

[0038] The results are shown in Table 1: Table 1 Performance of the self-made organic nitrogen source

[0039] The results show that when using wet bacterial residue alone, due to the presence of some colloidal substances, a hard blocky epidermis is formed after direct drying, and the moisture inside is still difficult to meet the standard, and it is difficult to sterilize and is prone to contamination, such as Comparative Example 1 and Comparative Example 3. When the proportion of mixed soybean powder is low, there is still agglomeration, such as in Example 3 where the mixing ratio of bacterial residue to soybean powder is 1:1.5, and the passing rate through a 60-mesh sieve is only 85%, resulting in a certain loss. Therefore, the ratio of soybean powder to bacterial residue should be at least greater than 1.5. In terms of moisture control, when the moisture is higher than 12%, contamination will occur, such as Comparative Example 5. If the moisture control is too low, there will be a waste of energy consumption. Therefore, the process of self-made organic nitrogen source is finally determined: the ratio of bacterial residue to soybean powder is 1:1.5 - 5, and the moisture is controlled at 5 - 10%. This can achieve the purpose of economical preparation and meeting the use effect.

[0040] Examples 7 - 12, Comparative Examples 6 - 9 Take part or all of the self-made organic nitrogen source prepared in Example 2 to replace soybean cake powder, configure the fermentation basal medium, and the formula and fermentation method are the same as in Example 1. The titer and finished product quality after fermentation are counted, and the results are shown in Table 2: Table 2 Influence of nitrogen source on gentamicin fermentation

[0041] The results show that the self-made organic nitrogen source can replace soybean powder for fermentation, and the product quality is qualified. Increasing the proportion of the self-made organic nitrogen source used in the fermentation basal material can improve the yield to a certain extent. If too much self-made bacterial residue with a large amount of bacterial residue is used, it will also cause contamination during fermentation; if the dry matter of the self-made organic nitrogen source is increased excessively, the increase in yield during fermentation is not proportional to the energy consumption, such as in Comparative Example 9, which may instead increase the cost.

[0042] Examples 13 - 16, Comparative Examples 11 - 12 Resource utilization of neomycin fermentation solid residue. Use the neomycin fermentation solid residue to prepare a self-made organic nitrogen source, and then conduct secondary fermentation of neomycin. The specific steps are as follows: (1) Obtain the fermentation solid residue by the same fermentation method as in Example 1. The formula of the fermentation basal medium consists of the following raw materials in weight percentages: 3% soybean cake powder, 6% rice flour, 0.3% corn steep liquor, 1.5% oral sugar, 0.5% ammonium sulfate, 0.5% NaCl, 0.5% light calcium carbonate, 0.03% KH 2 PO 4 ,0.06% Na 2 HPO 4 ,0.03% α - amylase, 0.2% silicone oil; (2) Mix the fermentation solid residue with 2 times the weight of soybean cake powder, stir evenly, then pass through a 20 - mesh sieve, and then dry at 45°C for 15 h, turning once every 3 h during the drying process to prepare a self-made organic nitrogen source with a water content of 7%. (3) Take self-made organic nitrogen sources with different contents for neomycin fermentation.

[0043] Table 3 Effects of nitrogen sources on neomycin fermentation

[0044] The results show that it is feasible to use the self-made organic nitrogen source to replace soybean meal in neomycin fermentation, and the dosage of soybean meal can be reduced. Increasing the self-made organic nitrogen source can increase the yield, and the product quality is qualified after replacement.

[0045] Examples 17 - 20, Comparative Examples 13 - 14 Resource utilization of apramycin fermentation solid residue. Prepare self-made organic nitrogen source from apramycin fermentation solid residue, and then carry out secondary fermentation of apramycin. The specific steps are as follows: (1) The fermentation method is the same as that in Example 1 to obtain fermentation solid residue. The formula of the fermentation basal medium consists of the following raw materials in weight percentages: 6% soybean cake powder, 4% oral glucose, 1% peptone, 0.5% light calcium carbonate, 0.5% ammonium chloride, 0.3% magnesium sulfate, 0.003% zinc sulfate heptahydrate, 2.5% corn flour, 0.4% corn steep liquor, 0.25% soybean oil, 0.03% potassium nitrate, 0.003% α - amylase, 0.1% antifoam (silicone oil); (2) Mix the fermentation solid residue with 2 times the weight of soybean cake powder, stir evenly, then pass through a 20 - mesh sieve, and then dry at 45°C for 15 h, turning once every 3 h during the drying process to prepare a self-made organic nitrogen source with a water content of 7%; (3) Take self-made organic nitrogen sources with different contents for neomycin fermentation.

[0046] Table 4 Effects of nitrogen sources on apramycin fermentation

[0047] The results show that using the self-made organic nitrogen source can relatively reduce the dosage of soybean cake powder. After replacing soybean cake powder for fermentation, the titer and quality are not affected and can be used as a substitute.

[0048] Example 21 Take the fermentation solid residues produced in Examples 9, 16 and 20, re - prepare self-made organic nitrogen source for fermentation, repeat multiple times, and observe the titer and impurity conditions after each fermentation end. The results are shown in Table 5.

[0049] Table 5 Effects of fermentation times on fermentation results

[0050] The results showed that when the self-made organic nitrogen source prepared from the bacterial residue was reused multiple times in fermentation, there was no obvious change in the yield and quality; indicating that multiple fermentations had no impact on the application of the fermentation solid residue, and the self-made organic nitrogen source could be repeatedly prepared for multiple fermentations to achieve long-term recycling.

[0051] Example 22 (1) Take the adsorption fermentation waste liquid in step (4) of Example 1, add the flocculant mother liquor according to a volume ratio of 16:0.1, stir evenly and let stand for 20 min, and then perform solid-liquid separation through a scraper centrifuge and a disc centrifuge to obtain the fermentation solid residue; the flocculant mother liquor is a PAM solution with a concentration of 1‰. (2) Mix the fermentation solid residue with 2 times the weight of soybean cake powder, stir evenly, pass through a 20-mesh sieve, and dry at 45°C for 15 h to obtain a self-made organic nitrogen source with a water content of 7%, and turn it over every 3 h during drying. (3) Use the self-made organic nitrogen source to prepare fermentation basal media respectively for the fermentation of apramycin, neomycin, and gentamicin.

[0052] Table 6 Influence of Flocculant on Fermentation

[0053] Comparative Example 15 The method and steps are the same as those in Example 22, only changing the volume ratio of the fermentation adsorption waste liquid to the flocculant mother liquor in step (1) to 9:1. It was found that too much flocculant was added to the fermentation adsorption waste liquid, and normal fermentation could not be carried out. The strains showed deformities, the bacterial concentration was low, and the fermentation titer was too low.

[0054] It can be seen from Example 22 and Comparative Example 15 that by appropriately adding the flocculant mother liquor to the adsorption fermentation waste liquid, a self-made organic nitrogen source can still be prepared, which will not affect the antibiotic fermentation process, and can save the usage amount of the flocculant to avoid excessive flocculant affecting normal fermentation.

Claims

1. A method for resource utilization of aminoglycoside antibiotic fermentation solid residue, characterized in that: The steps include: (1) Collecting fermentation solid residue: The adsorption fermentation waste liquid of aminoglycoside antibiotics is subjected to solid-liquid separation to obtain fermentation solid residue; (2) The fermentation solid residue is mixed with soybean cake powder, sieved, and dried to obtain a homemade organic nitrogen source; (3) Substituting homemade organic nitrogen source for soybean cake powder to prepare the fermentation basal medium; (4) Inoculate the seed liquid into the fermentation basal medium, ferment and extract to obtain aminoglycoside antibiotics.

2. The method for resource utilization of aminoglycoside antibiotic fermentation solid residue according to claim 1, characterized in that: The aminoglycoside antibiotic in step (1) is any one of apramycin, gentamicin and neomycin.

3. The method for resource utilization of aminoglycoside antibiotic fermentation solid residue according to claim 1, characterized in that: The solid-liquid separation method in step (1) is scraper centrifugation and disc centrifugation.

4. The method for resource utilization of aminoglycoside antibiotic fermentation solid residue according to claim 1, characterized in that: The water content of the fermentation solid residue in step (1) is 40-50%.

5. The method for resource utilization of aminoglycoside antibiotic fermentation solid residue according to claim 1, characterized in that: The mass ratio of the fermented solid residue to the soybean cake powder in step (2) is 1:1-5.

6. The method for resource utilization of aminoglycoside antibiotic fermentation solid residue according to claim 1, characterized in that: The sieve in step (2) is 20 mesh, and the drying condition is drying at 40-60° C. for 12-20 hours, and turning the mixture every 3 hours during drying.

7. The method for resource utilization of aminoglycoside antibiotic fermentation solid residue according to claim 1, characterized in that: The water content of the homemade organic nitrogen source in step (2) is 5-10%.

8. The method for resource utilization of aminoglycoside antibiotic fermentation solid residue according to claim 1, characterized in that: The amount of the homemade organic nitrogen source used in step (3) to replace soybean cake powder is 90-100% of the amount of soybean cake powder.

9. The method for resource utilization of aminoglycoside antibiotic fermentation solid residue according to claim 1, characterized in that: The volume ratio of the seed liquid to the fermentation basal medium in step (4) is 1:8-10.

10. The method for resource utilization of aminoglycoside antibiotic fermentation solid residue according to claim 1, characterized in that: The fermentation conditions in step (4) are as follows: a tank temperature of 36±0.5°C, an air volume of 0-55 L / min, and a rotation speed of 200-500 rpm.

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