CN108977365A

CN108977365A - The application of penicillium oxalicum L5 and its lincomycin fungi residues of degrading

Info

Publication number: CN108977365A
Application number: CN201810930453.2A
Authority: CN
Inventors: 李永红; 付路平; 韩正; 李晓静; 刘颖颖; 许福兰
Original assignee: Henan Daodi Biotechnology Co ltd
Current assignee: Zhengzhou University
Priority date: 2018-08-15
Filing date: 2018-08-15
Publication date: 2018-12-11
Anticipated expiration: 2038-08-15
Also published as: CN108977365B

Abstract

The invention discloses the applications of a kind of penicillium oxalicum L5 and its lincomycin fungi residues of degrading, the method of the application are as follows: the fermentation liquid that the fermented culture of penicillium oxalicum L5 obtains is seeded in lincomycin fungi residues, 25-37 DEG C, 80-220rpm culture, realize bacteria residue degradation.Penicillium oxalicum L5 of the present invention can be used for directly solving lincomycin fungi residues, be reached for 88.20% to the consumption rate of lincomycin fungi residues within six days, reduce harm of the bacteria residue to ecological environment and human health.

Description

The application of penicillium oxalicum L5 and its lincomycin fungi residues of degrading

Technical field

The present invention relates to the bacterial strains and method of a kind of harmless treatment lincomycin fungi residues, are carried out after especially handling Recycling recycles, and belongs to field of environment protection.

Background technique

China is antibiotics production big country, and main species antibiotic is produced per year more than 140,000 tons, produced during antibiotics production Raw solid waste (mycelium and protein) i.e. antibiotic bacterium dregs, annual output are more than 1,400,000 tons.

Antibiotic bacterium dregs are the solid fermentation wastes generated during fermenting and producing antibiotic, and main component is anti- The raw element mycelium of producing strains, the culture medium not utilized, the metabolite generated in fermentation process, culture medium degradation product with And a small amount of antibiotic etc., it is a kind of special hazardous waste, it is such as mishandling, ecological environment and human health can be generated Potentially hazardous, harm has the characteristics that concealment, hysteresis quality, cumulative, concertedness and related property.

Since containing a small amount of remaining antibiotic and its degradation product, to ecological environment, there is potential in antibiotic bacterium dregs Harmfulness.Environment-Ecosystem is made of in the form of food chain the biologic(al)group of different genera.The food animal such as livestock and poultry is long Phase low dosage takes in antibiotic, will lead to livestock and poultry and generates drug resistance to antibiotic, meanwhile, antibiotic is accumulated in animal body, is led It causes to generate antibiotic residue in animal foodstuff meat, egg, milk and internal organ.Antibiotic in animal foodstuff is transmitted to people along food chain, On the one hand it can cause crowd's allergic reaction, cause crowd to poison by food when serious；There are also carcinogenic, teratogenesis, mutagenesis for some drugs The effects of, severe jamming mankind's items physiological function.On the other hand, the animal foodstuff containing antibiotic can in human body intestinal canal just Normal flora generates adverse effect, destroys ecosystem balance in enteron aisle, makes pathogenic bacteria mass propagation；It can also be by drug-fast bacteria in animal The mankind are passed to, human health is threatened.Simultaneously because the bacteria residue content of organic matter is higher, secondary fermentation can be caused, color blackening produces Raw foul smell, seriously affects environment, and antibiotic bacterium dregs have been considered as one of the main public hazards of antibiotics production by international community, this It is the main reason for antibiotic production of raw medicine is transferred to third world countries by some developed countries in the world.

China's " pharmaceuticals industry pollution prevention technique policy " (bulletin 2012 No. 18) regulation antibiotic bacterium dregs are by danger Waste processing, forbids for producing feed.For the problems such as antibiotic bacterium dregs yield is big, processing difficulty is big, and " pharmacy work Industry pollution prevention technique policy " in propose " encourage reutilization technology of the exploitation fermentation bacteria residue in production technology, innoxious place Reason technology, comprehensive utilization technique " policy and suggestion seek a kind of economic, efficient and big treating capacity method, to realize antibiotic bacterium Slag is rationally and efficiently used to be of great significance with safe disposal.

Include at present burning to the processing disposal technology of antibiotic bacterium dregs, Fertilizer Transformed (compost), fodder (being prohibited), fill out Bury, energy (producing methane through anaerobic fermentation) and other processing disposal technologies.The main composition of bacteria residue is moisture, and drying needs to disappear A large amount of energy is consumed, and the calorific value of bacteria residue is not high；Many antibiotic bacterium dregs contain the elements such as more sulphur and nitrogen, increase in burning The desulfurization burden and the pollution of oxynitrides of boiler are added.Antibiotic residue problem that may be present causes Fertilizer Transformed not big Crowd receives.Since soil is in short supply, landfill is not long-term plan.The complexity of antibiotic bacterium slag ingredient increases its solid fermentation Difficulty；Remaining antibiotic inhibits the fermentation growth of microorganism in bacteria residue；Bacteria residue corruption is fast, and the solid fermentation time is longer, hair Stink is serious during ferment, generates secondary pollution.Bacteria residue is used as the thinking that fermentation nitrogen source is another recycling.

Remaining antibiotic can inhibit the growth of microorganism in bacteria residue, therefore, this is decomposed and is conducive to its subsequent applications. Report about lincomycin degradation has two: one plant clostridiums through co-culturing 10 days with bacteria residue, for 100 mg/L lincomycins Degradation rate is 62.03%, and if initial concentration is 500 mg/L, degradation rate only has 15.61%；One plant of geotrichum candidum is trained altogether with bacteria residue After supporting 10 days, the degradation rate to lincomycin is 37 %.

Antibiotic fermentation gives up in bacteria residue, due to having a small amount of remaining antibiotic and its degradation product, to ecological environment there is Potentially hazardous property is considered as one of the main public hazards of antibiotics production by international community.Big for antibiotic bacterium dregs yield, What is proposed in the problems such as processing difficulty is big, and " pharmaceuticals industry pollution prevention technique policy " " encourages exploitation fermentation bacteria residue in life Reutilization technology, innoxious process for treating, comprehensive utilization technique in production. art " policy and suggestion, seek it is a kind of it is economical, efficiently and The big method for the treatment of capacity is of great significance with realizing that antibiotic bacterium dregs are rationally and efficiently used with safe disposal.

It include at present burning, Fertilizer Transformed (compost), landfill, energy to the processing disposition common technology of antibiotic bacterium dregs (producing methane through anaerobic fermentation) and other processing disposal technologies have its shortcoming.

Summary of the invention

The present invention provides a kind of new strains of lincomycin fungi residues of degrading -- penicillium oxalicum (Penicillium oxalicum) L5 and its it is innoxious degradation lincomycin fungi residues in application.

The specific technical solution of the present invention is as follows:

In a first aspect, the present invention provides one plant of new strains -- penicillium oxalicum (Penicillium oxalicum) L5, it is preserved in State's Microbiological Culture Collection administration committee common micro-organisms center, preservation date on April 8th, 2018, deposit number CGMCC No.15400, address Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.

Second aspect, the present invention provide a kind of application of penicillium oxalicum L5 in degradation lincomycin fungi residues, the application Method are as follows: by penicillium oxalicum L5 it is fermented culture obtain fermentation liquid be seeded in lincomycin fungi residues, 25-37 DEG C culture (preferably 28 DEG C) realize bacteria residue degradation.

Further, the fermentating liquid volume dosage is calculated as 0.1 ~ 10 mL/g, preferably 0.5ml/ with lincomycin fungi residues weight g；Mycelium content is 1~5 g/mL, preferably 3.5g/mL in the fermentation liquid.

Further, the fermentation liquid is prepared as follows: penicillium oxalicum L5 is seeded to potato culture medium, and (i.e. fermentation is trained Support base), in 20~37 DEG C, preferably 28 DEG C of 80~220 r/min fermented and cultured, 36~54 h(, 200 r/min, 48 h)；It is described Potato culture medium composition: 200 g/L of potato, 20 g/L of glucose, 20 g/L of agar, solvent are deionized water, and pH value is certainly So.

Further, inclined-plane and seed culture are first carried out before the penicillium oxalicum L5 fermentation, then by seed liquor with volumetric concentration 2 The inoculum concentration of ~ 10%(preferably 10%) is seeded to fermentation medium: penicillium oxalicum L5 is seeded to slant medium, 20~37 DEG C of cultures Preferably 37 DEG C of 12~72 h(cultures 48 h), obtain inclined-plane thalline；The slant medium quality final concentration composition are as follows: glucose 0.5~2%, peptone 0.2~2%, beef extract 0.1~2%, sodium chloride 0.1~2%, agar 2%, solvent are distilled water, pH value 6~ 7.5；Inclined-plane thalline is seeded to seed culture medium again, cultivates 12~72 h(preferably 37 in 20~37 DEG C, 80~220 r/min DEG C, 180 r/min culture 48 h), obtain seed liquor；The seed culture medium final concentration composition are as follows: glucose 0.5~2%, egg White peptone 0.2~2%, beef extract 0.1~2%, sodium chloride 0.5~2%, solvent are distilled water, pH value 6~7.5.

Further, slant medium (i.e. ordinary broth agar medium) the final concentration composition: glucose 0.5%, egg White peptone 1%, beef extract 0.1%, NaCl 0.5%, agar 2%, solvent are distilled water, pH value 7.3-7.5.

Further, seed culture medium (i.e. broth medium) the final concentration composition: glucose 0.5%, peptone 1%, beef extract 0.1%, NaCl 0.5%, solvent is distilled water, pH value 7.3-7.5.

Further, content of lincomycin is 5 ~ 20 mg/g, lincomycin fungi of the present invention in the lincomycin fungi residues Slag is mainly from Henan pharmaceutcal corporation, Ltd of old name for the Arabian countries in the Middle East.

In lincomycin fungi residues degradation process of the present invention, can by way of feed supplement lincomycin fungi residues into Row degradation, feed supplement bacteria residue add equivalent and bacteria residue, can at most add 5 times after having degraded.

Compared with prior art, beneficial effect of the present invention is mainly reflected in:

The present invention provides a kind of lincomycin fungi residues degradation method, mainly using penicillium oxalicum (Penicillium oxalicum) L5, it can be used for directly solving lincomycin fungi residues, 88.20% be reached for the consumption rate of lincomycin fungi residues in six days, Reduce harm of the bacteria residue to ecological environment and human health.We have found directly to degrade for the first time micro- lifes of antibiotic bacterium dregs Object had no such report in the past.

Detailed description of the invention

Fig. 1 is the growth curve of bacterial strain.

Fig. 2 lincomycin fungi residues degradation amount change curve.

Specific embodiment

For the present invention is better described, special as follows for embodiment, but the scope of the present invention is not limited thereto.

Broth medium quality final concentration composition: glucose 0.5%, peptone 1%, 0.1% beef extract, 0.5% chlorination Sodium, solvent are deionized water, pH value 7.4 ± 0.1.

Ordinary broth agar medium quality final concentration composition: glucose 0.5%, peptone 1%, 0.1% beef extract, 0.5% Sodium chloride, agar 2%, solvent are deionized water, pH value 7.4 ± 0.1.

YEPD (one) culture medium quality final concentration composition: glucose 2%, yeast extract 1%, peptone 2%, solvent are deionization Water, pH are natural.

YEPD (two) culture medium quality final concentration composition: glucose 2%, peptone 2%, solvent are deionized water, and pH is certainly So.

Potato culture medium: 200 g/L of potato, 20 g/L of glucose, 20 g/L of agar, solvent are deionized water, pH It is worth nature.

1 bacteria selection of embodiment and identification

1, bacterial strain screening

Broth agar culture medium quality final concentration composition: peptone 1%, beef extract 0.3%, sodium chloride 0.5%, agar 2%, solvent are Deionized water, pH value 7.4.

The dilution of sewage different multiples (respectively 2.5 that will be obtained in the aerobic tank of medicine company sewage treatment plant of Henan old name for the Arabian countries in the Middle East Again, 25 times, 250 times, 2500 times and 25000 times), lincomycin is added, making lincomycin concentration is respectively 0.6 g/L and 6 g/ L.It is configured to a series of sewage lincomycin mixture.

It pours into 20 mL broth agar culture mediums while hot in each culture dish, plate is made.It then can by above-mentioned sewage woods Mycin mixture is applied on plate, in 37 DEG C of 12 h of culture.According to colony characteristics, two kinds of bacterial strains are filtered out, are denoted as bacterium respectively Strain L5 and bacterial strain L6.

2, bacterial strain is identified

1) colony characteristics

The cellular morphology of bacterial strain L5 are as follows: irregular elongated tubular product has bifurcated.

The cellular morphology of bacterial strain L6 are as follows: have branch, the elongated tubular to cross one another.

Judge L5~L6 for mould according to the microscopy results of colony characteristics.

2) characterization of molecules

(1) mycelial preparation

Thallus after purification is inoculated in broth medium, 28 DEG C of 36~48 h of culture.To mycelial growth to diameter It is best when 4 mm or more (larger than mung bean smaller than general soya bean).

(2) mycelial separation and preservation

The mycelium prepared is moved into 10 mL centrifuge tubes, -20 DEG C of 24 h of freezen protective.The specific operation method is as follows: taking 5 mL liquid transfer gun heads (will guarantee to suck mycelium and not leak outside) to cut, be inhaled with this pipette tips with scissors at 6 mm of pipette tips front end Bacterium solution is taken to shift into centrifuge tube.Centrifuge tube equipped with bacterium solution is put into a centrifuge into 5000 r/min and is centrifuged 20 min, in abandoning Clear liquid.As for the additional amount of bacterium solution, mycelium height is near 1 mL graduation mark of centrifuge tube after guaranteeing centrifugation.If primary Find that the mycelia scale of construction is insufficient, can be added again bacterium solution into the centrifuge tube for discard supernatant liquid and be centrifuged again, until mycelia after centrifugation Until body is enough.

(3) ultrasonic method extracts DNA

Taking-up fills the mycelial centrifuge tube of freezing, and 5 mL sterile waters are added and wait for that defrosting (can heat, not exceed 40 DEG C) after shake up, be put into 10 min of ultrasonic vibration in Ultrasound Instrument.5000 r/min are centrifuged 10 min after the completion of concussion, abandon supernatant Benzyl chloride Extraction buffer (pH=9.0) 3 mL, 20% SDS solution, 0.4 mL, 2.4 mL of benzyl chloride stoste is added in liquid.It fills Concussion is divided to shake up, 50 DEG C of 1 h of water-bath to 2 h shake up once every 10 min, are completely dissolved and (are seen after 1 h of water-bath after mycelium Mycelium dissolved state is examined, if difference not can be improved temperature water-bath 1 is h) again to 60 DEG C before dissolved state and water-bath very much.It is added Sodium acetate solution (pH=5.2) 2.4 mL, 15 min of ice bath.5000 r/min are centrifuged 15 min after ice bath.Take supernatant 4 4 mL of isopropanol is added into new centrifuge tube in mL, 5000 r/min centrifugation 10 after 20 min of precipitation at room temperature after careful mixing min.Supernatant is abandoned, twice with 80% ethanol washing precipitating, by ethyl alcohol drying (to keep the wet of precipitating) in super-clean bench.Add Enter 300 1 × TE of uL weight molten DNA precipitating, when dissolution shakes up light.So far DNA slightly proposes completion.

It takes 200 uL slightly to mention DNA in 1.5 mL centrifuge tubes, isometric phenol/chloroform/isoamyl alcohol is added, gently up and down 30 times reverse, using refrigeration centrifuge, 12000 r/min are centrifuged 5 min under the conditions of 18 DEG C, take 100 uL of supernatant in new In 1.5 mL centrifuge tubes.So far fungal DNA, which extracts, completes.

4.PCR amplification and electrophoresis detection

Using 18S rRNA common template NS1(5 '-GTAGTCATATGCTTGTCTC-3 ') and NS2(5 '- GGCTGCTGGCACCAGACTTGC-3 ') DNA of fungi is expanded.

20 μ L of PCR amplification system: each 0.5 μ L of upstream and downstream primer, 0.5 ng/ of μ L(10~100 of DNA profiling of extraction μ L), sterilized 8.5 μ L of ultrapure water is eventually adding 2 × Ex Tap enzyme, 10 μ L.

The operating parameter of PCR amplification program: the operating parameter of PCR amplification program:

(94 DEG C, 5 min)+(94 DEG C, 30 s)+(55 DEG C, 30 s)+(72 DEG C, 1 min) } × 30+(72 ℃, 7 min)。

The electrophoresis detection of PCR product after amplification: expanding the PCR product of end, uses 1 × TE as the slow of electrophoresis Fliud flushing carries out electrophoresis, deposition condition using 1% Ago-Gel are as follows: 150 V of voltage, 20 min of time are clapped after electrophoresis According to being analyzed using particular analysis software.Hangzhou Jin Weizhi company is sent to be sequenced PCR stoste (no less than 10 μ L).

Identify that bacterial strain L5(18 sRNA nucleotide sequence is as shown in SEQ ID NO.1 through 18 sRNA) it is penicillium oxalicum (Penicillium oxalicum)；Bacterial strain L6(18 sRNA nucleotide sequence is as shown in SEQ ID NO.2) it is penicillium chrysogenum (Penicillium chrysogenum).

The foundation of 2 lincomycin standard curve of embodiment

Stationary phase: phase chromatography-use octadecylsilane chemically bonded silica (5 μm) column, the mm of 4.6 mm × 200, mobile phase: 0.05 The borax soln (adjusting pH value to 6.1 with 85% phosphoric acid solution) of M and methanol mixing (volume ratio 42:58), flow velocity: 1 mL/ min.Column temperature: 30 DEG C, detector: UV detector, Detection wavelength: 214 nm.

Under the above conditions, content of lincomycin (x) and UV absorption (peak area, y) within the scope of 0~8.0 mg/mL Linear, equation of linear regression is y=1.1013x -0.0203, R²= 0.9997。

3 Spawn incubation of embodiment

Bacterial strain L5, bacterial strain L6 that embodiment 1 is filtered out are seeded to ordinary broth agar medium respectively, 37 DEG C of cultures are laggard Row slant preservation is in case use later；Slant strains are accessed in broth medium, is sealed with 8 layers of gauze, is put into shaking table In at 37 DEG C 200 r/min cultivate 48 h, obtain seed.

The selection of 4 fermentation medium of embodiment

Because L5 is mould, it is respectively connected in broth medium and potato culture medium and (does multiple parallel tests), in 28 DEG C, 200 r/min culture, periodically remove one bottle, weigh to mycelia.It the results are shown in Table 1, discovery potato culture culture is imitated Fruit is preferable, and bacterium ball grows fast.

Potato culture medium prescription: 200 g/L of potato, 20 g/L of glucose, 20 g/L of agar, solvent is deionization Water, pH value are natural.Bottled 100 mL of liquid of 500 mL triangle of liquid amount.

Table 1 cultivates the dry mycelial weight (g) of the L5 of different time

Incubation time (h)	Broth medium	Potato culture medium
			12	0.07	0.09
24	0.12	0.17
			36	0.18	0.29
48	0.23	0.38
			60	0.23	0.39

It was found that growing preferable with potato culture medium culture thallus.Growth curve is shown in Fig. 1

5 bacteria residue Degrading experiment of embodiment

Bacteria residue is derived from Henan pharmaceutcal corporation, Ltd of old name for the Arabian countries in the Middle East, is the solid waste by generating after the hydraulic filter of lincomycin fermentation, Lin Ke Mycin content is 16.77 mg/g.

(1) 4 the method for embodiment is pressed, respectively the slant strains of switching bacterial strain L5 and bacterial strain L6 to broth medium In, 37 DEG C, 48 h are grown under the conditions of 180 r/min；Seed liquor is inoculated into potato culture with the inoculum concentration of volumetric concentration 10% again In base, in 28 DEG C, 200 r/min cultivate 48 h, obtain fermentation liquid, and wet hyphae content is 3.5 g/mL.

(2) 5 mL of bacterium solution for taking (1) two kind of mould of step respectively is uniformly transferred to 10 sterilized g lincomycin fungis On slag；It takes 5 mL sterile waters to be uniformly connected on same processed bacteria residue simultaneously and does blank control；

(3) being arranged 6 days is a cycle, daily sampling weighing, calculates bacteria residue consumption rate.Bacteria residue consumption rate calculation formula:

Consumption rate=(M0-Mx) ÷ M0 × 100%(M0 is bacteria residue weight before experiment starts, and Mx is xth day bacteria residue weight)

Bacterial strain L5 and bacterial strain L6, the visible amount by culture bacteria residue of naked eyes significantly reduces, using decrement weight method to two plants of moulds The effect of consumption lincomycin fungi residues is measured.Find that two plants of moulds were in the 6th day consumption lincomycin fungi residues by measurement Consumption rate be respectively 88.20% and 75.60%, significant effect (degradation curve Fig. 2).Wherein bacterial strain L5 produce sour mould effect compared with It is good, it is sent to China Committee for Culture Collection of Microorganisms's common micro-organisms center and carries out preservation, deposit number CGMCC No.15400。

6 bacteria residue of embodiment adds test

5 mL embodiments 4 are added in the bacteria residue described in 10 g embodiments 5, and using the culture of potato culture medium, to stationary phase, (3.5 g are wet Mycelia/mL) L5 bacterium solution, cultivated in 28 DEG C, after the bacteria residue in culture dish is complete by microbial consumption, be added 10 g it is new Bacteria residue cultivated again, as follows repeatedly 5 times.It was found that adding 5 bacteria residues can be consumed.

Sequence table

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Claims

1. penicillium oxalicum (Penicillium oxalicum) L5, it is common to be preserved in China Committee for Culture Collection of Microorganisms Microorganism center, preservation date on April 8th, 2018, deposit number CGMCC No.15400, address: the Chaoyang District, Beijing City North Star The institute 3 of West Road 1.

2. a kind of application of claim 1 penicillium oxalicum L5 in degradation lincomycin fungi residues.

3. application as claimed in claim 2, it is characterised in that the method for the application are as follows: by the fermented culture of penicillium oxalicum L5 The fermentation liquid of acquisition is seeded in lincomycin fungi residues, and bacteria residue degradation is realized in 25~37 DEG C of cultures.

4. application as claimed in claim 3, it is characterised in that the fermentating liquid volume dosage is in terms of lincomycin fungi residues weight For 0.1 ~ 10 mL/g.

5. application as claimed in claim 3, it is characterised in that the fermentation liquid is prepared as follows: penicillium oxalicum L5 is connect Kind obtains fermentation liquid in 20~37 DEG C, 80~220 r/min fermented and cultured, 36~54 h to fermentation medium；The fermentation training Base composition: 200 g/L of potato, 20 g/L of glucose, 20 g/L of agar is supported, solvent is deionized water, and pH value is natural.

6. application as claimed in claim 4, it is characterised in that first carry out inclined-plane and seed training before the penicillium oxalicum L5 fermentation It supports, then seed liquor is seeded to fermentation medium with the inoculum concentration of volumetric concentration 2 ~ 10%: penicillium oxalicum L5 is seeded to inclined-plane training Base is supported, 20~37 DEG C of 12~72 h of culture obtain inclined-plane thalline；The slant medium quality final concentration composition are as follows: glucose 0.5~2%, peptone 0.2~2%, beef extract 0.1~2%, sodium chloride 0.1~2%, agar 2%, solvent are distilled water, pH value 6~ 7.5；Inclined-plane thalline is seeded to seed culture medium again, 12~72 h is cultivated in 20~37 DEG C, 80~220 r/min, is planted Sub- liquid；The seed culture medium final concentration composition are as follows: glucose 0.5~2%, peptone 0.2~2%, beef extract 0.1~2%, chlorine Change sodium 0.5~2%, solvent is distilled water, pH value 6~7.5.

7. application as claimed in claim 6, it is characterised in that the slant medium final concentration composition: glucose 0.5%, egg White peptone 1%, beef extract 0.1%, NaCl 0.5%, agar 2%, solvent are distilled water, pH value 6~7.5.

8. application as claimed in claim 6, it is characterised in that the seed culture medium final concentration composition: glucose 0.5%, egg White peptone 1%, beef extract 0.1%, NaCl 0.5%, solvent are distilled water, pH value 6~7.5.

9. application as claimed in claim 3, it is characterised in that content of lincomycin is 0.2~2 in the lincomycin fungi residues mg/g。

10. application as claimed in claim 3, it is characterised in that mycelium content is 1 ~ 5 g/mL in the fermentation liquid.