CN108977365B

CN108977365B - Penicillium oxalicum L5 and application thereof in degradation of lincomycin bacterial residues

Info

Publication number: CN108977365B
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: Zhengzhou University
Current assignee: Zhengzhou University
Priority date: 2018-08-15
Filing date: 2018-08-15
Publication date: 2021-08-27
Anticipated expiration: 2038-08-15
Also published as: CN108977365A

Abstract

The invention discloses a penicillium oxalicum L5 and application thereof in degrading lincomycin bacterial residues, wherein the application method comprises the following steps: and (3) inoculating fermentation liquor obtained by fermenting and culturing penicillium oxalicum L5 into lincomycin bacterial residues, and culturing at 25-37 ℃ and 80-220rpm to realize bacterial residue degradation. The penicillium oxalicum L5 can be used for directly decomposing lincomycin bacterial residues, the consumption rate of the lincomycin bacterial residues for six days reaches 88.20%, and the harm of the bacterial residues to the ecological environment and the human health is reduced.

Description

Penicillium oxalicum L5 and application thereof in degradation of lincomycin bacterial residues

Technical Field

The invention relates to a strain and a method for harmlessly treating lincomycin bacterial residues, in particular to a strain which is treated and then recycled, and belongs to the field of environmental protection.

Background

China is a big antibiotic producing country, the annual production of main antibiotics exceeds 14 million tons, and the annual production of solid wastes (mycelium and protein), namely antibiotic fungi residues, produced in the antibiotic producing process exceeds 140 million tons.

The antibiotic residues are solid fermentation wastes generated in the process of producing antibiotics by fermentation, the main components of the antibiotic residues are mycelium of antibiotic producing bacteria, unused culture medium, metabolites generated in the fermentation process, degradation products of the culture medium, a small amount of antibiotics and the like, the antibiotic residues are special dangerous wastes, and if the antibiotic residues are not properly treated, the antibiotic residues can generate potential hazards to the ecological environment and the human health, and the hazards have the characteristics of concealment, hysteresis, accumulation, cooperativity, continuity and the like.

The antibiotic residues contain a small amount of residual antibiotics and degradation products thereof, so that the antibiotic residues are potentially harmful to the ecological environment. The environmental ecosystem is composed of different species of biota in the form of a food chain. The long-term low-dose intake of antibiotics by food animals such as livestock and poultry can cause the livestock and poultry to generate drug resistance to the antibiotics, and meanwhile, the antibiotics accumulate in the bodies of the animals to cause the antibiotic residues in meat, eggs, milk and internal organs of the animal food. Antibiotics in animal food are transmitted to people along a food chain, on one hand, allergic reaction of people can be caused, and in severe cases, food poisoning of people can be caused; some medicines also have the effects of carcinogenesis, teratogenesis, mutagenesis and the like, and seriously interfere various physiological functions of human beings. On the other hand, animal food containing antibiotics can have adverse effects on normal flora in intestinal tracts of human bodies, destroy balance of ecosystem in the intestinal tracts and enable pathogenic bacteria to propagate in a large quantity; but also can transfer drug-resistant bacteria in animals to human beings, thus threatening human health. Meanwhile, the organic matter content of the fungus dregs is high, secondary fermentation can be caused, the color becomes black, foul smell is generated, and the environment is seriously influenced.

The antibiotic fungi residues are treated according to dangerous waste and prohibited to be used for producing feed according to the regulation of pharmaceutical industry pollution prevention and treatment technical policy (No. 18 in 2012) in China. Aiming at the problems of large production amount and large treatment difficulty of antibiotic fungi residues and the policy suggestion of ' encouraging the development of reuse technology, harmless treatment technology and comprehensive utilization technology of fermented fungi residues in the production process ' in the technical policy of pollution prevention and control in pharmaceutical industry ', an economic, efficient and large-treatment-capacity method is sought, so that the reasonable and effective utilization and safe treatment of the antibiotic fungi residues are realized, and the method has important significance.

The current treatment and disposal technologies for antibiotic fungi residues comprise incineration, composting, forage (prohibited), landfill, energy regeneration (anaerobic fermentation for producing methane) and other treatment and disposal technologies. The main composition of the mushroom dregs is water, a large amount of energy is consumed for drying the mushroom dregs, and the heat value of the mushroom dregs is not high; many antibiotic fungi residues contain more elements such as sulfur, nitrogen and the like, and increase the desulfurization burden of a boiler and the pollution of oxynitride during combustion. The possible problem of antibiotic residues renders the composting unacceptable to the public. Because of the shortage of land, landfill is not a long-term measure. The complexity of the components of the antibiotic fungi residues increases the difficulty of solid fermentation; the residual antibiotics in the mushroom dregs inhibit the growth of the microorganisms for fermentation; the fungus dregs are decomposed quickly, the solid fermentation time is long, the odor is serious in the fermentation process, and secondary pollution is generated. The mushroom dregs are used as a fermentation nitrogen source, which is another resource-recycling idea.

The residual antibiotic in the mushroom residue inhibits the growth of microorganisms, and therefore, degrades this to facilitate subsequent use thereof. There are two reports on lincomycin degradation: the degradation rate of the clostridium strain is 62.03 percent for 100 mg/L lincomycin after the clostridium strain is cultured with the mushroom dregs for 10 days, and the degradation rate is only 15.61 percent if the initial concentration is 500 mg/L; after the geotrichum candidum and the mushroom dregs are cultured for 10 days, the degradation rate of the lincomycin is 37%.

The antibiotic fermentation waste residues have potential hazard to the ecological environment due to a small amount of residual antibiotics and degradation products thereof, and have been regarded as one of the main public hazards of antibiotic production by the international society. Aiming at the problems of large production amount and large treatment difficulty of antibiotic fungi residues and the policy suggestion of ' encouraging the development of reuse technology, harmless treatment technology and comprehensive utilization technology of fermented fungi residues in the production process ' in the technical policy of pollution prevention and control in pharmaceutical industry ', an economic, efficient and large-treatment-capacity method is sought, so that the reasonable and effective utilization and safe treatment of the antibiotic fungi residues are realized, and the method has important significance.

At present, common treatment technologies for antibiotic bacteria residues comprise incineration, fertilizer formation (composting), landfill, energy regeneration (anaerobic fermentation for producing methane) and other treatment technologies, which have the defects.

Disclosure of Invention

The present invention provides a new bacterial strain-penicillium oxalicum (B) for degrading lincomycin bacterial dregsPenicillium oxalicum) L5 and application thereof in harmless degradation of lincomycin bacterial residues.

The specific technical scheme of the invention is as follows:

in a first aspect, the invention provides a novel strainStrain-penicillium oxalicum (B)Penicillium oxalicum) L5, deposited in China general microbiological culture Collection center, with the date of 2018, 4 and 8 months, the preservation number CGMCC No.15400, and No. 3, Xilu No.1, North Chen, of the Chaojing area, Beijing, the address.

In a second aspect, the invention provides an application of penicillium oxalicum L5 in degradation of lincomycin bacterial residues, and the application method comprises the following steps: and (3) inoculating fermentation liquor obtained by fermenting and culturing penicillium oxalicum L5 into lincomycin bacterial residues, and culturing at 25-37 ℃ (preferably 28 ℃) to realize bacterial residue degradation.

Further, the volume usage amount of the fermentation liquid is 0.1-10 mL/g, preferably 0.5mL/g, based on the weight of the lincomycin bacterial residues; the content of mycelia in the fermentation liquid is 1-5 g/mL, and 3.5g/mL is preferred.

Further, the fermentation liquor is prepared by the following method: inoculating penicillium oxalicum L5 to a potato culture medium (namely a fermentation culture medium), and carrying out fermentation culture at 20-37 ℃ and 80-220 r/min for 36-54 h (preferably 28 ℃, 200 r/min and 48 h); the potato culture medium comprises the following components: 200 g/L of potato, 20 g/L of glucose, 20 g/L of agar and deionized water as a solvent, and the pH value is natural.

Further, before fermentation, the penicillium oxalicum L5 is subjected to slant culture and seed culture, and then the seed solution is inoculated to a fermentation medium in an inoculum size of 2-10% (preferably 10%) by volume: inoculating penicillium oxalicum L5 to a slant culture medium, and culturing at 20-37 ℃ for 12-72 h (preferably at 37 ℃ for 48 h) to obtain slant bacteria; the final concentration of the slant culture medium comprises the following components by mass: 0.5-2% of glucose, 0.2-2% of peptone, 0.1-2% of beef extract, 0.1-2% of sodium chloride and 2% of agar, wherein the solvent is distilled water, and the pH value is 6-7.5; inoculating the slant thalli to a seed culture medium, and culturing at 20-37 ℃ and 80-220 r/min for 12-72 h (preferably culturing at 37 ℃ and 180 r/min for 48 h) to obtain a seed solution; the final concentration of the seed culture medium is as follows: 0.5-2% of glucose, 0.2-2% of peptone, 0.1-2% of beef extract, 0.5-2% of sodium chloride and distilled water as a solvent, wherein the pH value is 6-7.5.

Further, the final concentration composition of the slant medium (i.e., common broth agar medium): 0.5 percent of glucose, 1 percent of peptone, 0.1 percent of beef extract, 0.5 percent of NaCl, 2 percent of agar and distilled water as a solvent, and the pH value is 7.3-7.5.

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

Furthermore, the lincomycin content in the lincomycin bacterial residues is 5-20 mg/g, and the lincomycin bacterial residues are mainly from Henan Tianfang pharmaceutical industry Co.

In the process of degrading the lincomycin bacterial residues, the lincomycin bacterial residues can be degraded in a mode of supplementing materials and adding the lincomycin bacterial residues, and the same amount of the lincomycin bacterial residues are added after the lincomycin bacterial residues are degraded, and the lincomycin bacterial residues can be added for 5 times at most.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a method for degrading lincomycin bacterial residues, which mainly utilizes penicillium oxalicum (penicillium oxalicum)) (Penicillium oxalicum) L5, can be used for directly dissolving lincomycin bacterial residues, has a consumption rate of 88.20 percent for six days, and reduces the harm of the bacterial residues to the ecological environment and the human health. We have found for the first time microorganisms that can directly degrade antibiotic residues, and such reports have not been seen before.

Drawings

FIG. 1 is a growth curve of the strain.

FIG. 2 shows the change curve of the degradation amount of lincomycin bacterial residues.

Detailed Description

For better illustration of the present invention, the following specific examples are given, but the scope of the present invention is not limited thereto.

The quality final concentration composition of a common broth culture medium is as follows: 0.5% of glucose, 1% of peptone, 0.1% of beef extract, 0.5% of sodium chloride and deionized water as a solvent, wherein the pH value is 7.4 +/-0.1.

The composition of the final concentration of the common broth agar medium is as follows: 0.5% of glucose, 1% of peptone, 0.1% of beef extract, 0.5% of sodium chloride, 2% of agar and deionized water as a solvent, wherein the pH value is 7.4 +/-0.1.

YEPD (I) culture medium quality final concentration composition: 2% of glucose, 1% of yeast extract, 2% of peptone and deionized water as a solvent, wherein the pH is natural.

YEPD (II) culture medium quality final concentration composition: 2% of glucose, 2% of peptone and deionized water as a solvent, and the pH is natural.

Potato culture medium: 200 g/L of potato, 20 g/L of glucose, 20 g/L of agar and deionized water as a solvent, and the pH value is natural.

Example 1 screening and identification of bacterial species

1. Strain screening

The final concentration composition of the broth agar medium is as follows: peptone 1%, beef extract 0.3%, sodium chloride 0.5%, agar 2%, solvent deionized water, and pH 7.4.

Diluting sewage obtained from aerobic pond of pharmaceutical industry sewage treatment plant in Henan Tian Fang by different times (2.5 times, 25 times, 250 times, 2500 times and 25000 times respectively), and adding lincomycin to make lincomycin concentration be 0.6 g/L and 6 g/L respectively. Preparing a series of lincomycin mixtures for sewage.

20 mL of broth agar was poured into each dish while it was hot, and the plate was prepared. The lincomycin mixture was then spread onto plates and incubated at 37 ℃ for 12 h. Two strains were selected according to colony characteristics and were designated strain L5 and strain L6, respectively.

2. Identification of strains

1) Characteristics of bacterial colony

The cell morphology of strain L5 was: irregular slender tube shape with bifurcations.

The cell morphology of strain L6 was: has branched, long tubular shape intersecting with each other.

According to the microscopic observation result of the colony characteristics, L5-L6 are judged to be the mold.

2) Molecular characteristics

(1) Preparation of mycelia

And inoculating the purified bacteria into a common broth culture medium, and culturing at 28 ℃ for 36-48 h. It is best when the mycelium grows to a diameter of more than 4 mm (larger than mung bean and smaller than general soybean).

(2) Isolation and preservation of mycelia

The prepared mycelia were transferred into a 10 mL centrifuge tube and stored frozen at-20 ℃ for 24 hours. The specific operation method comprises the following steps: a5 mL pipette tip is taken, the position 6 mm away from the front end of the pipette tip (ensuring that mycelia can be sucked in and do not leak) is cut off by scissors, and the pipette tip is used for sucking the bacterial liquid and transferring the bacterial liquid into a centrifuge tube. And (4) placing the centrifugal tube filled with the bacterial liquid into a centrifugal machine for centrifugation at 5000 r/min for 20 min, and discarding the supernatant. And (3) ensuring that the height of the centrifuged mycelium is close to the 1 mL scale mark of the centrifuge tube according to the addition amount of the bacteria liquid. If the mycelium amount is insufficient after one-time centrifugation, adding the bacterial liquid into the centrifuge tube with the supernatant discarded again, and centrifuging again until enough mycelium exists.

(3) Ultrasonic method for extracting DNA

Taking out the centrifuge tube containing the frozen mycelia, adding 5mL of sterile water, thawing (heating at 40 deg.C or below), shaking, and placing into an ultrasonic instrument for ultrasonic oscillation for 10 min. After shaking, centrifugation is carried out for 10 min at 5000 r/min, the supernatant is discarded, and 3 mL of benzyl chloride extraction buffer (pH =9.0), 0.4 mL of 20% SDS solution and 2.4 mL of benzyl chloride stock solution are added. Shaking thoroughly, water bath at 50 deg.C for 1-2 h, shaking once every 10 min until the mycelium is completely dissolved (observing the dissolution state of mycelium after water bath for 1 h, and if the dissolution state and the water bath front region are not large enough, increasing the temperature to 60 deg.C and then water bath for 1 h). Sodium acetate solution (pH =5.2) 2.4 mL was added and ice-cooled for 15 min. Centrifuging at 5000 r/min for 15 min after ice bath. Taking 4 mL of the supernatant to a new centrifuge tube, adding 4 mL of isopropanol, carefully mixing uniformly, precipitating at room temperature for 20 min, and centrifuging at 5000 r/min for 10 min. The supernatant was discarded, the pellet was washed twice with 80% ethanol, and the ethanol was blown dry in a clean bench (to keep the pellet wet). The DNA precipitate was redissolved by the addition of 300 uL of 1 XTE, which was gently shaken during the dissolution. This completes the crude extraction of DNA.

And (3) putting 200 uL of the crude DNA into a 1.5 mL centrifuge tube, adding phenol/chloroform/isoamylol with the same volume, slightly reversing the mixture up and down for 30 times, centrifuging the mixture for 5 min at 12000 r/min at 18 ℃ by using a refrigerated centrifuge, and taking 100 uL of supernate into a new 1.5 mL centrifuge tube. This completes the extraction of fungal DNA.

PCR amplification and electrophoresis detection

The DNA of the fungus was amplified using 18S rRNA universal templates NS1 (5'-GTAGTCATATGCTTGTCTC-3') and NS2 (5'-GGCTGCTGGCACCAGACTTGC-3').

PCR amplification system 20. mu.L: 0.5. mu.L of each of the upstream and downstream primers, 0.5. mu.L (10 to 100 ng/. mu.L) of the extracted DNA template, 8.5. mu.L of sterilized ultrapure water, and finally 10. mu.L of 2 XEx Tap enzyme were added.

Operating parameters of the PCR amplification program: operating parameters of the PCR amplification program:

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

and (3) carrying out electrophoretic detection on the amplified PCR product: the amplified PCR product was electrophoresed on 1% agarose gel using 1 XTE as the electrophoresis buffer under the following conditions: voltage 150V, time 20 min, taking pictures after electrophoresis, and analyzing by using specific analysis software. The PCR stock solution (not less than 10. mu.L) was sent to Hangzhou Jinzhi company for sequencing.

Identified by 18 sRNA, the strain L5 (the 18 sRNA nucleotide sequence is shown in SEQ ID NO. 1) is penicillium oxalicum: (Penicillium oxalicum) (ii) a The strain L6 (18 sRNA nucleotide sequence is shown in SEQ ID NO. 2) is penicillium chrysogenum (A)Penicillium chrysogenum）。

EXAMPLE 2 establishment of a lincomycin Standard Curve

Stationary phase: octadecylsilane chemically bonded silica (5 μm) column for liquid chromatography, 4.6 mm × 200 mm, mobile phase: 0.05M borax solution (pH adjusted to 6.1 with 85% phosphoric acid solution) and methanol were mixed (volume ratio 42: 58) at a flow rate: 1 mL/min. Column temperature: 30 ℃, detector: ultraviolet detector, detection wavelength: 214 nm.

Under the conditions, the lincomycin content (x) and the ultraviolet absorption (peak area, y) are in a linear relation within the range of 0-8.0 mg/mL, and the linear regression equation is y = 1.1013 x-0.0203, R²= 0.9997。

EXAMPLE 3 Strain culture

Respectively inoculating the strain L5 and the strain L6 selected in the example 1 into a common broth agar culture medium, culturing at 37 ℃, and performing slant preservation for later use; inoculating slant strain into common broth culture medium, sealing with 8 layers of gauze, and culturing in shaking table at 37 deg.C for 48 h at 200 r/min to obtain seed.

Example 4 selection of fermentation Medium

Since L5 is a mold, it was inoculated into a common broth medium and a potato medium, respectively (for multiple parallel experiments), cultured at 28 ℃ at 200 r/min, one bottle was periodically removed, and the mycelia were weighed. The results are shown in Table 1, and it is found that the culture effect is better when potato is used for culture, and the growth of the fungus balls is fast.

The potato culture medium formula comprises: 200 g/L of potato, 20 g/L of glucose, 20 g/L of agar and deionized water as a solvent, and the pH value is natural. The liquid loading volume is 500 mL and the liquid loading volume is 100 mL.

TABLE 1 Dry weight (g) of mycelia of L5 cultured for various periods

Incubation time (h)	General broth culture 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 the cells were grown well by culturing them in a potato medium. The growth curve is shown in FIG. 1.

Example 5 fungal dreg degradation test

The mushroom dregs are obtained from Henan Tian prescription pharmaceutical industry Co., Ltd, and are solid wastes generated by filter pressing of lincomycin fermentation liquor, and the lincomycin content is 16.77 mg/g.

(1) Slant strains of strain L5 and strain L6 were transferred to a common broth and grown at 37 ℃ and 180 r/min for 48 hours as described in example 4; inoculating the seed solution into a potato culture medium according to the inoculation amount with the volume concentration of 10%, and culturing at 28 ℃ at 200 r/min for 48 h to obtain a fermentation liquid with the wet mycelium content of 3.5 g/mL.

(2) Respectively taking 5mL of the bacterial liquid of the two moulds in the step (1), and uniformly transferring the bacterial liquid to 10 g of sterilized lincomycin residues; meanwhile, 5mL of sterile water is uniformly connected to similarly treated mushroom dregs to be used as blank control;

(3) setting 6 days as a period, sampling and weighing every day, and calculating the bacteria dreg consumption rate. The bacteria dreg consumption rate calculation formula is as follows:

consumption rate = (M0-Mx) ÷ M0 × 100% (M0 is the weight of the mushroom residue before the start of the experiment, Mx is the weight of the mushroom residue on day x)

The bacterial strain L5 and the bacterial strain L6 can obviously reduce the amount of the cultured bacterial residues by naked eyes, and the effect of the two moulds consuming the lincomycin bacterial residues is measured by adopting a decrement weighing method. According to the determination, the consumption rates of the two moulds consuming the lincomycin residues on the sixth day are respectively 88.20% and 75.60%, and the effect is obvious (degradation curve chart 2). The strain L5 has good effect on producing acid penicillium, and is delivered to the China general microbiological culture Collection center for preservation, with the preservation number of CGMCC No. 15400.

Example 6 fungi residue supplement test

To 10 g of the fungal residue described in example 5, 5mL of the L5 bacterial solution of example 4 cultured in potato medium to a stationary phase (3.5 g wet mycelia/mL) was added, and the mixture was cultured at 28 ℃ and, after the fungal residue in the dish was consumed by the microorganisms, 10 g of fresh fungal residue was added and the culture was repeated 5 times. The mushroom dregs were found to be consumed after 5 additional times.

Sequence listing

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Claims

1. Penicillium oxalicum (B)Penicillium oxalicum) L5, deposited in China general microbiological culture Collection center with the date of 20184, 8 days, preservation number CGMCC No.15400, address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North.

2. Use of the penicillium oxalicum L5 of claim 1 for degrading lincomycin bacterial residues.

3. The application according to claim 2, characterized in that the method of application is: and (3) inoculating fermentation liquor obtained by fermenting and culturing penicillium oxalicum L5 into lincomycin bacterial residues, and culturing at 25-37 ℃ to realize bacterial residue degradation.

4. The use according to claim 3, wherein the volume of the fermentation liquid is 0.1-10 mL/g based on the weight of the lincomycin residues.

5. Use according to claim 3, characterized in that the fermentation broth is prepared as follows: inoculating penicillium oxalicum L5 to a fermentation culture medium, and carrying out fermentation culture at 20-37 ℃ at a speed of 80-220 r/min for 36-54 h to obtain a fermentation liquid; the fermentation medium comprises the following components: 200 g/L of potato, 20 g/L of glucose, 20 g/L of agar and deionized water as a solvent, and the pH value is natural.

6. The use of claim 4, wherein the Penicillium oxalicum L5 is cultured by slant culture and seed culture before fermentation, and the seed solution is inoculated into the fermentation medium in an inoculum size of 2-10% by volume: inoculating penicillium oxalicum L5 to a slant culture medium, and culturing at 20-37 ℃ for 12-72 h to obtain slant bacteria; the final concentration of the slant culture medium comprises the following components by mass: 0.5-2% of glucose, 0.2-2% of peptone, 0.1-2% of beef extract, 0.1-2% of sodium chloride and 2% of agar, wherein the solvent is distilled water, and the pH value is 6-7.5; inoculating the slant thalli to a seed culture medium, and culturing at 20-37 ℃ and 80-220 r/min for 12-72 h to obtain a seed solution; the final concentration of the seed culture medium is as follows: 0.5-2% of glucose, 0.2-2% of peptone, 0.1-2% of beef extract, 0.5-2% of sodium chloride and distilled water as a solvent, wherein the pH value is 6-7.5.

7. The use according to claim 6, wherein the final concentration of the slant medium consists of: 0.5% of glucose, 1% of peptone, 0.1% of beef extract, 0.5% of NaCl, 2% of agar and distilled water as a solvent, wherein the pH value is 6-7.5.

8. Use according to claim 6, characterized in that the final concentration composition of the seed culture medium is: 0.5% of glucose, 1% of peptone, 0.1% of beef extract, 0.5% of NaCl and distilled water as a solvent, wherein the pH value is 6-7.5.

9. The use according to claim 3, wherein the lincomycin content in the lincomycin bacterial residue is 0.2-2 mg/g.

10. The use according to claim 3, wherein the mycelium content in the fermentation broth is 1 to 5 g/mL.