CN103173508A - Method for cleaning and producing lincomycin - Google Patents
Method for cleaning and producing lincomycin Download PDFInfo
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- CN103173508A CN103173508A CN 201110437782 CN201110437782A CN103173508A CN 103173508 A CN103173508 A CN 103173508A CN 201110437782 CN201110437782 CN 201110437782 CN 201110437782 A CN201110437782 A CN 201110437782A CN 103173508 A CN103173508 A CN 103173508A
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Abstract
The invention provides a novel method for cleaning and producing lincomycin. The method is characterized in that a lincomycin producing strain is adopted and used for fermenting at a ventilation condition; the obtained fermentation liquor is acidified and then filtered; the filtered clear liquid is alkalinized and then extracted by mixed alcohol; the extract liquid is centrifuged to separate, wherein the upper organic phase is used for extracting the lincomycin, and the lower aqueous phase is the producing wastewater which can be used as one of the components of fermentation mediums of the next batch after being subjected to component analysis, thus realizing the recycle of the producing wastewater. According to the method for cleaning and producing the lincomycin, the effective components in the producing wastewater are fully utilized, the purpose of turning wastes into wealth is realized, so that the waste of the resource, as well as the pollution to the environment, are reduced; the production cost is decreased, and the pressure for environmental protection due to the drainage of the producing waste is relieved at the same time.
Description
Technical field
The present invention relates to a kind of production method of biological raw material medicine, specifically a kind of by the novel method of microorganism fermentative production lincomycin.
Background technology
Lincomycin, the industrial U-10149 that claims again, be within 1962, by Mason, from streptomycete, to cultivate species to separate a kind of lincosamides obtained, component comprises amino acid and sugar.Lincomycin has good restraining effect to gram-positive microorganism, and it does not have the toxicity of cross resistance and itself lower, so be widely used in the disease that gram-positive microorganism causes, Gram-negative bacteria is also had to certain restraining effect simultaneously.Lincomycin distributes extensively in vivo, can enter marrow and osseous tissue, and the clinical treatment that is widely used in the illnesss such as respiratory system, osteomyelitis and soft tissue infection, be particularly useful for the patient to penicillin and erythromycin allergy.Lincomycin is also the starting raw material as clindamycin, Clindamycin Phosphate, clindamycin palmitate etc. in addition.
Just because of the widespread use of lincomycin and the huge market requirement, manufacturer is numerous at present, turnout is huge, Nanyang Pu Kang according to statistics, Henan old name for the Arabian countries in the Middle East, Anhui Province north, year production that the North China pharmacy is four is just more than 5000 tons, yet because current Global Environmental Problems becomes increasingly conspicuous, the wastewater problem of factory also more and more receives people's concern, the main source of the waste water of the production of lincomycin is the lower floor's factory effluent after the organic solvent extraction layering, be rich in the phosphorus (> 40mg/L of high density in waste water), nitrogen (amino nitrogen content > 60mg/100ml) and a large amount of inorganic ion.Although in a large amount of relevant patent of lincomycin of application before, the production wastewater treatment that relates to lincomycin is arranged, as Chinese patent CN1865175A(treating agent for lincomycin hydrochloride production waste water and preparation method thereof and using method), the CN102079607A(waste water advanced processing of lincomycin and middle water can use technique) be all to concentrate on how to carry out the wastewater treatment aspect, cure the symptoms, not the disease.If Efficient Cycle utilizes factory effluent in process of production, will when improving raw material availability, can reduce processing cost and the quantity discharged of factory effluent, reach the purpose of lincomycin cleaner production.
Summary of the invention
The pollution problem of the waste water produced for lincomycin fermentation to environment, the invention provides a kind of novel method of lincomycin cleaner production.
Technical scheme of the present invention: a kind of production method of lincomycin, comprise seed culture, fermentating culturing process, the alcohol mixture extraction process, it is characterized in that, fermented liquid through lincomycin producing strain fermentation gained, through the laggard andante frame of oxalic acid acidifying, filter, adopt the alcohol mixture extraction after crossing the cleaner liquid alkalization, after the extraction liquid layering, lower floor's water supplements appropriate carbon source after composition analysis, nitrogenous source, inorganic ion and water, form the circulating fermentation substratum, and make circulating fermentation medium component and initial medium component content and index quite or equate, the circulating fermentation substratum is through adjusting pH, carry out circulating fermentation after sterilizing and produce lincomycin, thereby be built into the clean preparation method of a lincomycin circulating fermentation.
Described fermented liquid is 1～6 through the pH of oxalic acid acidifying scope, is preferably 3～3.5.
Cross basifier that the cleaner liquid alkalization used for one or more in sodium carbonate, sodium bicarbonate, sodium hydroxide, potassium hydroxide after the fermented liquid acidifying, alkalization pH scope is 8～12, is preferably 10～11.
The C such as the n-Octanol that described alcohol mixture is two or more, nonylcarbinol
7～C
11monohydroxy-alcohol, preferably n-Octanol and nonylcarbinol, blending ratio is n-Octanol: nonylcarbinol (v/v)=5:1～1:5.
Described fermentation initial medium comprises following composition (by weight percentage): starch saccharificating liquid 5～20%, soybean cake powder 0.1～10%, corn steep liquor 0.1～5%, ammonium sulfate 0.1～3%, calcium carbonate 0.3～3%, sodium-chlor 0.1～1%, potassium primary phosphate 0.01～0.15%, surplus is water, pH6.5～8.0.
After after described extraction liquid layering, the reducing sugar of lower floor's waste water phase, amino nitrogen, total phosphorus and sodium-chlor index are analyzed, supplement appropriate saccharified liquid, soybean cake powder, corn steep liquor, ammonium sulfate, calcium carbonate, sodium-chlor, potassium primary phosphate and process water, adjust pH6.5～8.0, form the circulating fermentation substratum, make circulating fermentation medium component and initial medium component content and index quite or equate.Wherein supplementary saccharification liquid measure be in initial fermention medium saccharified liquid 70～95%, ammonium sulfate be in initial fermention medium ammonium sulfate 10～40%, potassium primary phosphate be in initial fermention medium potassium primary phosphate 20～50%, sodium-chlor be in initial fermention medium sodium-chlor 20～50%.
Fermentation strain used be the streptomyces lincolnensis lincomycin producing strain (
streptomyces lincolnensis) the CPCC-200038 bacterial strain, therefrom traditional Chinese medicines are bought with microbial strains preservation administrative center.
The sterilization process condition of circulating fermentation substratum is intermittently sterilizing in batches in 110～130 ℃, 20～40 minutes.
When the present invention adopts the circulating fermentation substratum to carry out aerobic fermentation, fermentation condition is: inoculum size is 5%～15%(v/v), 25～35 ℃ of leavening temperatures, ventilation is 0.8～2.0vvm, tank pressure 0.01～0.06MPa, mixing speed is 100～500rpm, fermentation time is 170～208h.
Beneficial effect of the present invention: lincomycin product of the present invention is pharmaceutical grade, in the rear leaching process of lincomycin, utilize product to reach the principle of the different solubility in different solvents under condition of different pH, adopt organic solvent extraction, produce a large amount of factory effluents after extracting and demixing, be rich in the compositions such as abundant nitrogen, phosphorus, inorganic salt in this factory effluent, if directly discharge causes serious environmental problem, and the processing of factory effluent will increase the production cost of lincomycin greatly.The present invention is grouped into and is analyzed by the one-tenth to factory effluent, factory effluent is included in the formula of fermention medium, build the utilization process of Lincomycin wastewater, cycle index is 3～8 times, the quantity discharged of factory effluent is reduced to 10～30% of former technique, the water consumption of fermenting process substratum preparation is reduced to 40～60% of former technique, reduces the consumption of raw material saccharified liquid, ammonium sulfate, potassium primary phosphate, sodium-chlor, can reduce production costs.
The accompanying drawing explanation
Fig. 1: the production technological process of lincomycin
Embodiment
Below in conjunction with specific embodiment, the invention will be further described.Should be understood that following examples are only for the present invention is described but not for limiting the scope of the invention.
In the following example, for indicating the experimental technique of actual conditions, the condition of usually recommending according to industry normal condition or production firm is carried out.
Except special instruction, the % used in following examples, ‰ is weight ratio.
Embodiment 1: the preparation of lincomycin seed liquor
Lincomycin producing strain be the streptomyces lincolnensis lincomycin producing strain (
streptomyces lincolnensis) the CPCC-200038 bacterial strain
Slant medium: starch 2%, sodium-chlor 0.05%, saltpetre 0.5%, dipotassium hydrogen phosphate 0.1%, sal epsom 0.05%, ferrous sulfate 0.01 ‰, agar 1.8%, surplus is water, the pH nature.
Seed culture medium: starch 1.5%, glucose 3%, soybean cake powder 1.5%, corn steep liquor 2.5%, SODIUMNITRATE 0.2%, ammonium sulfate 0.2%, potassium primary phosphate 0.05%, calcium carbonate 0.5%, soya-bean oil 0.05%, bubble enemy 0.01%, surplus is water, pH value: before 7.6～7.8(disappears).
Female inclined-plane seed preparation: in sterilisable chamber, get 1/5 shovel sand spore inoculating under aseptic technique in through cultivating on one day aseptic normal test tube slant, smoothen, under 29 ℃ of relative humidity 45% conditions, cultivate 6～7 days, select aseptic inclined-plane normal, that spore is plentiful, outward appearance is good standby in 1-4 ℃ of Refrigerator store, can preserve one month.
Sub-inclined-plane seed preparation: in sterilisable chamber, get above-mentioned slant pore under aseptic technique, with transfering loop scraping one ring spore, be uniformly coated on test tube slant, under the culture condition identical with female inclined-plane, cultivate 5～6 days, this inclined-plane can save backup in 1-4 ℃ of refrigerator, preservation period 10-15 days.
The preparation of bacteria suspension: in sterilisable chamber, get the cultured test tube slant every pipe of seed under aseptic technique and add the 5ml stroke-physiological saline solution, scrape spore, vibrate gently and obtain bacteria suspension, the multitube bacteria suspension is transferred in inoculation bottle, standby as the inoculation seeding tank.
The seed liquor culture condition:
Inoculum size: bacteria suspension prepared with 5 test tubes by every 1L seed liquor, temperature: 29～31 ℃, process pH between 6.4～6.8, incubation time 46～50h, tank pressure 0.04mpa.
Seed liquor culture transferring index:
It is light grey that the mycelia outward appearance is, and the thalline microscopy is Shu Tuanzhuan, and body is thick, stretches effectively, and dyeing is dark, pH > 6.7, reducing sugar (%)<1.0, bacterium dense (v/v): 30～40%.
Embodiment 2: the preparation of lincomycin fermentation liquid
Initial fermention medium: starch saccharificating liquid 6.5%, soybean cake powder 2%, corn steep liquor 1.5%, ammonium sulfate 0.38%, calcium carbonate 0.8%, sodium-chlor 0.68%, potassium primary phosphate 0.03%, surplus is water, pH6.5～8.0.
Supplemented medium 1: soybean cake powder 10%, corn steep liquor 18%, calcium carbonate 0.8%, soya-bean oil 0.05Kg/m
3, bubble enemy 0.01 Kg/m
3, surplus is water, the pH nature.
Supplemented medium 2: soybean cake powder 4%, corn steep liquor 6%, SODIUMNITRATE 2%, ammonium sulfate 1%, potassium primary phosphate 0.05%, calcium carbonate 0.5%, soya-bean oil 0.05Kg/m
3, bubble enemy 0.05 Kg/m
3, surplus is water, the pH nature.
Fermentation condition:
Fermenting process is implemented in the fermentor tank of 50L, initial loading liquid measure 50%, inoculum size is 6%, mixing speed 300rpm, ventilation 1.2vvm, tank pressure 0.04mpa, 30 ± 1 ℃ of leavening temperatures, fermentation time is 180～208h, before fermentation, 40h samples every 4h, after 40h, every the 8h sampling, the sample determination index comprises reducing sugar, amino nitrogen, total phosphorus, bacterium dense (%), tires and repetition measurement pH.In fermenting process, the measure of control such as feed supplement and pH are as follows:
Reducing sugar: start to mend sugar after inoculation after reducing sugar is down to below 1.2%, adopt 36% saccharified liquid controlled fermentation before 100h reducing sugar amount 1.2%, after fermentation 100h reducing sugar 0.8%, put tank before 10h stop mending sugar.
PH: after before fermentation, 100h is controlled at 7.2～7.4,100h by logical ammonia, pH is controlled at 6.8～7.2, wherein ammonia concn > 20%.
Amino nitrogen: whole process is controlled at 30～40mg/100ml, adopts ammoniacal liquor (when fermented liquid pH is on the low side) or 10% ammonium sulfate (when fermented liquid pH is higher) to regulate the amino nitrogen level.
Feed supplement: respectively at 32h, 60h, disposable 2.5L, 1.5L, 1L, the 1L supplemented medium 1 of filling into of 110h, 140h after the fermentor tank inoculation, the disposable 2.5 L supplemented mediums 2 that fill into of 90h after the fermentor tank inoculation.
Embodiment 3: the product preparation of lincomycin
The about 40L of fermented liquid by gained in embodiment 2, adopt the oxalic acid pH that will ferment to be adjusted to 3.0, acidizing fluid adopts Plate Filtration, must cross cleaner liquid 22L, cross cleaner liquid and adopt 20% sodium hydroxide solution to adjust pH to 10.5, alkaline filtrate carries out multiple filter and guarantees the alkali lye clarification, adds the alcohol mixture of 2/5 volume, start and stir 20-30min, static layering.Upper organic phase obtains the lincomycin product after the unit operations such as vacuum concentration, crystallization, decolouring, acetone crystallization, drying.
Embodiment 4: the preparation of the recycle of factory effluent and circulating fermentation substratum
Get alcohol mixture extraction liquid lower floor factory effluent in embodiment 3 and carry out composition analysis, result is as following table:
Table 1: Contents of Main Components in factory effluent
Adopt factory effluent as one of circulating fermentation medium component, carry out the preparation of circulating fermentation substratum according to the component content in factory effluent: the treatment process that embodiment 3 states obtains the 20L factory effluent, starch saccharificating liquid 1503.0g, soybean cake powder 500.0g, corn steep liquor 375.0g, ammonium sulfate 11.1g, calcium carbonate 200.0g, sodium-chlor 52.0g, potassium primary phosphate 3.2g, process water 4L, adjust pH6.5～8.0.
By fermention medium consumption 25L, according to the described treatment process of embodiment 3, obtain the 20L factory effluent, during the preparation of process for cleanly preparing circulating fermentation substratum, the ratio of factory effluent and process water is about 5:1, during recycle 8 times, the output of lincomycin and component B content of lincomycin relative standard deviation are all in 5%, show that circulating fermentation does not affect output and the quality of lincomycin substantially, can reduce about 150L discharge of wastewater through 8 circulating fermentations (50L fermentor tank lab scale level), result is as shown in the table:
Table 2: circulating fermentation experimental result
| Cycle index | Lincomycin yield (U/ml) | Lincomycin B content (%) |
| 0 | 5823 | 3.04 |
| 1 | 5689 | 3.19 |
| 2 | 5968 | 2.91 |
| 3 | 5981 | 2.89 |
| 4 | 5788 | 3.01 |
| 5 | 5862 | 3.05 |
| 6 | 5911 | 3.00 |
| 7 | 5793 | 3.10 |
| 8 | 5839 | 3.05 |
Claims (2)
1. the production method of a lincomycin, comprise seed culture, fermentating culturing process, the alcohol mixture extraction process, it is characterized in that, fermented liquid through lincomycin producing strain fermentation gained, through the laggard andante frame of oxalic acid acidifying, filter, adopt the alcohol mixture extraction after crossing the cleaner liquid alkalization, after the extraction liquid layering, lower floor's water supplements appropriate carbon source after composition analysis, nitrogenous source, inorganic ion and water, form and ferment initial medium component content and index quite or the circulating fermentation substratum equated, the circulating fermentation substratum is through adjusting pH, carry out circulating fermentation after sterilizing and produce lincomycin, the initial medium that wherein ferments comprises following composition (by weight percentage): starch saccharificating liquid 5～10%, soybean cake powder 0.1～10%, corn steep liquor 0.1～5%, ammonium sulfate 0.1～3%, calcium carbonate 0.3～3%, sodium-chlor 0.1～1%, potassium primary phosphate 0.01～0.15%, bubble enemy 0.05～0.5 Kg/m
3, surplus is water, pH6.5～8.0.
2. method according to claim 1, after it is characterized in that the indexs such as total reducing sugar, amino nitrogen, total phosphorus and sodium-chlor of lower floor's waste water phase after the extraction liquid layering are analyzed, supplement appropriate saccharified liquid, soybean cake powder, corn steep liquor, ammonium sulfate, calcium carbonate, sodium-chlor, potassium primary phosphate and process water, adjust pH6.5～8.0, form the circulating fermentation substratum, make circulating fermentation medium component and initial medium component content and index quite or equate; Wherein supplementary saccharification liquid measure be in initial fermention medium saccharified liquid 70～95%, ammonium sulfate be in initial fermention medium ammonium sulfate 10～40%, potassium primary phosphate be in initial fermention medium potassium primary phosphate 20～50%, sodium-chlor be in initial fermention medium sodium-chlor 20～50%.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111592576A (en) * | 2020-05-11 | 2020-08-28 | 天方药业有限公司 | Treatment method of butanol crystallization mother liquor of lincomycin hydrochloride |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111592576A (en) * | 2020-05-11 | 2020-08-28 | 天方药业有限公司 | Treatment method of butanol crystallization mother liquor of lincomycin hydrochloride |
| CN111592576B (en) * | 2020-05-11 | 2023-11-24 | 天方药业有限公司 | Treatment method of butanol crystallization mother liquor of lincomycin hydrochloride |
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Application publication date: 20130626 |