CN101259313B - Method for degrading residual tylosin in medicine slag - Google Patents
Method for degrading residual tylosin in medicine slag Download PDFInfo
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- CN101259313B CN101259313B CN2008100933963A CN200810093396A CN101259313B CN 101259313 B CN101259313 B CN 101259313B CN 2008100933963 A CN2008100933963 A CN 2008100933963A CN 200810093396 A CN200810093396 A CN 200810093396A CN 101259313 B CN101259313 B CN 101259313B
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Abstract
The invention relates to a method for degrading residual tylosin in herb residues, in particular to a method for degrading residual tylosin in herb residues by utilizing microorganism, which mainly comprises the steps of preparation of soil fungi suspension, preparation of strain breeding cultural medium, preparation of strain domestication cultural medium, preparation of strain screening cultural medium, preparation of stain activation and rejuvenation cultural medium, preparation of drug residues fermentation cultural medium, screening of degrading strain and so on. The invention provides a compound bacterium that can be screened and cultured to degrade the residual tylosin in herb residues, while strains for degrading the residual tylosin in herb residues can be acquired by being separated and purified from the compound bacterium. Then the strains can be prepared to be corresponding herb residue cultural medium, which can degrade the residual tylosin in herb residues after being accessed the fermentation of the degrading bacteria.
Description
Technical field
The present invention relates to the method for residual tylosin in a kind of dregs of a decoction of degrading, especially relate to a kind of method of utilizing residual tylosin in the microbial degradation dregs of a decoction.
Background technology
Because antibiotic dregs of a decoction content of organic matter height is nutritious, simple airing or stacking very easily cause secondary fermentation, the color blackening, and produce frowst.Last century the nineties, people mainly concentrate on the treatment process of the antibiotic dregs of a decoction in the tackling key problem of dry technology.1991, the anti-medical group company in Shandong, Shandong antibiolics dregs dyring process was succeedd.1996, the anti-feed factory in Shandong, Shandong worked out centrifuge again in conjunction with fluidized bed drying penicillin wet slag, made drying cost reduce by 30%.
Along with succeeding in developing of various dregs of a decoction drying equipments, domestic have the man unit of number to carry out the antibiotic dregs of a decoction as high protein feed raw material and medicine Study on Additive.1998-1999, Li Yuehai etc. utilize anti-safe happy mycelian protein in Shandong and cephalo mycelian protein to replace the dregs of beans of equivalent to be used for laying hen with 1.5%, 3% addition respectively and raise, obtain feeding effect preferably; With tylosin protein feed feeding meat chick, fryer survival rate, speed of weight increment, feedstuff-meat ratio all are significantly higher than not interpolation group.
Yet the exploitation of the above-mentioned dregs of a decoction is used, and does not all eliminate antibiotic residual in the dregs of a decoction.Because after animal eats the dregs of a decoction of residual antibiotic for a long time, can produce all adverse consequences such as drug-fast bacteria, livestock products medicament residue.This just means the dregs of a decoction as the regenerative feedstuff resource, must at first degrade wherein residual antibiotic.
In recent years, the research report of residual antibiotic degradation technique is very few in the relevant dregs of a decoction both at home and abroad, but the report of the research aspect the residual antibiotic improvement is more in urban domestic wastewater and industrial wastewater.Tang Shaohong etc. directly are used for flowers with the form of fertilizer with the dregs of a decoction and use, and have also obtained satisfied fertilization effect, but residual in the dregs of a decoction antibiotic are arranged, and long-term use may have a negative impact to the soil micro-ecosystem balance.Studies show that: tetracycline can be accumulated in environment, and then destroys the microbial balance in the water and soil earth; Tylosin can cause that resistance salmonella quantity increases greatly in the environment.Kummerer etc., Ingerslev etc., Drillia etc. adopt bioanalysis, degradation technique to residual Ciprofloxacin, Ofloxacin, flagyl, SMZco and terramycin for animals etc. in the sanitary wastewater is studied, but resulting result is inconsistent.Main cause is: 1. different antibiotic, and its molecular structure and stability are different; 2. degrade and be still waiting further screening and optimize with bacterial strain; 3. inorganic in the sewage, organic different to different strains degraded different antibiotic influence possibility; 4. temperature, pH value, dissolved oxygen etc. are to the influence of different degradation processes.Lange etc., Andreozzi etc. carry out harmless treatment with ozone to macrolide antibiotics residual in the sewage such as erythromycin, CLA, ROX, lincomycin, obtained comparatively satisfied regulation effect, administer the research of still needing but whether this method is fit to the dregs of a decoction.Also the someone adopts the electrochemical oxidation method to handle antibiotic residue in the waste water, finds can effectively reduce Ofloxacin content in the waste water, but residual lincomycin is not had remarkable degradation, but this method is not suitable for the plant layout processing with the method, because of energy consumption too big.Otker etc., Polubesova etc. adsorb medicines such as residual tetracycline, Enrofloxacin in the sewage with porous type silicate minerals such as montmorillonite, zeolites, and then sewage is purified, but this method is not fundamentally eliminated residual antibiotic, be that change has taken place in the antibiotic site that exists, also be not suitable for the thorough improvement of residual antibiotic in the dregs of a decoction.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, provide a kind of by screening, cultivate the compound bacteria of residual tylosin in the dregs of a decoction of can degrading, and then from compound bacteria, separate, bacterial strain that purifying obtains degrading tylosin, prepare corresponding dregs of a decoction culture medium, insert residual tylosin in the degradation bacteria fermentative degradation dregs of a decoction.
The present invention realizes in the following way:
The method of residual tylosin in a kind of dregs of a decoction of degrading, it is characterized in that: this method comprises the steps:
1) preparation of soil bacteria suspension: use quartering, take by weighing and stack near the soil sample 1～10g of tylosin herb residue, place triangular flask, add 100～200ml PBS, after shaking up, place the constant temperature shaking table, under 25～35 ℃, the 200r/min 20～40min that vibrates;
2) strain improvement culture medium preparation: with the raw material of following weight mix get final product the strain improvement culture medium: tylosin herb residue 200g, peptone 10g, glucose 10g, K
2HPO
40.1g, FeSO
47H
2O 0.2g, water 800ml;
3) strain domestication culture medium preparation: with following raw material mix by weight get final product the strain domestication culture medium: 1. one-level domestication culture medium: tylosin herb residue and water are prepared in 1: 9 ratio; 2. secondary is tamed culture medium: tylosin herb residue and water are prepared in 2: 8 ratio; 3. tame culture mediums for three grades: tylosin herb residue and water are prepared in 3: 7 ratio; 4. level Four is tamed culture medium: tylosin herb residue and water are prepared in 4: 6 ratio;
4) bacterial screening culture medium preparation: with the raw material of following weight mix get final product the bacterial screening culture medium: 1. primary dcreening operation isolation medium: tylosin herb residue 200g, agar 20g, water 800ml; 2. multiple screening the: yeast extract powder 10g, peptone 20g, glucose 20g, Tylosin Tartrate standard items 0.04g, water 1000ml from culture medium; 3. separation and purification culture medium: peptone 10g, glucose 5g, tylosin herb residue leachate 1000ml;
5) actication of culture rejuvenation culture medium preparation: with the raw material of following weight mix get final product actication of culture rejuvenation culture medium: yeast extract powder 10g, peptone 10g, tylosin herb residue leachate 1000ml;
6) preparation of dregs of a decoction fermentation medium: with the raw material of following weight mix get final product dregs of a decoction fermentation medium: tylosin herb residue 700～900g, wheat bran 300～100g, K
2HPO
40.1～0.2g, FeSO
47H
2O 0.1～0.2g, water 800～900ml;
7) get above-mentioned steps 1) the soil bacteria suspension 10～20ml of preparation, join 100～200g above-mentioned steps 2) in the strain improvement culture medium of preparation, stir, putting in 25～35 ℃ of constant temperature shaking tables, 120r/min cultivates down 60～72h; Get the bacteria suspension after the cultivation, inoculum concentration by 2～5% is linked into above-mentioned steps 3) in the first class inoculum domestication culture medium of preparation, put in 25～35 ℃ of constant temperature shaking tables, 120r/min cultivates 60～72h, repeat this step 5～10 time, used strain domestication culture medium rises to level Four step by step by one-level;
8) getting nutrient solution 1～3ml after the domestication, is 10 with the PBS gradient dilution
-1, 10
-210
-10Concentration, adopt pour plate method will dilute back bacterium liquid then and be inoculated in the primary dcreening operation isolation medium, pour into a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices after the mixing, difference according to colonial morphology and growth cycle thereof, number consecutively also inserts liquid with it and sieves culture medium again, after cultivation is gone down to posterity, adopt the spread plate method to be inoculated into solid again and sieve culture medium again, through after the separation it being inserted fluid separation applications purifying culture medium once more, cultivation is gone down to posterity the back streak inoculation to solid separation and purification culture medium, and after repeating this step 3～4 time, finish the separation and purification of bacterial classification, can filter out the bacterial strain of degraded tylosin;
9) get above-mentioned steps 8) degraded tylosin bacterial strain, be linked into above-mentioned steps 5) preparation actication of culture rejuvenation culture medium in, put in 25～35 ℃ of constant temperature shaking tables, 120r/min cultivates 20～30h down, behind the centrifugal 15min of 4000r/min, be suspended in the new actication of culture rejuvenation culture medium, 120r/min continues to cultivate 8～14h in 25～35 ℃ of constant temperature shaking tables, can obtain tylosin degradation bacteria suspension;
10) get above-mentioned steps 6) the dregs of a decoction fermentation medium 100～200g of preparation, insert above-mentioned steps 9) the tylosin degradation bacteria suspension 2～10ml of preparation, and stirring, 25～35 ℃ of fermentation 60～120h 60～100 ℃ of oven dry down, get final product after the pulverizing then;
The tylosin herb residue leachate of described step 4) is that the ratio according to dregs of a decoction weight in wet base and water is preparation in 1: 2, and concrete steps are as follows:
A. get tylosin herb residue 100g, add sterilized distilled water of 200ml or deionized water, at room temperature soak 12～20h after stirring;
B. soak among the above-mentioned steps a is sub-packed in the centrifuge tube, gradient centrifugation 30min abandons precipitation;
C. get the supernatant after the gradient centrifugation, abandon floating impurity by suction filtration again and get final product;
The domestication process of described step 7) tylosin degradation bacteria has experienced two stages, and it is followed successively by stabilization sub stage of bacterial strain proterties and the bacterial strain stabilization sub stage to the tylosin degradation capability.
Tylosin herb residue is after said method is handled, and the tylosin content assaying method is as follows in the dregs of a decoction:
1) preparation of tylosin calibration curve
Compound concentration is respectively the Tylosin Tartrate standard liquid of 0 μ g/ml, 2 μ g/ml, 4 μ g/ml, 6 μ g/ml, 8 μ g/ml, 10 μ g/ml, 14 μ g/ml, 16 μ g/ml, 18 μ g/ml and 20 μ g/ml, getting 10 μ l point samples is S1 solid medium (the culture medium composition: peptone 8g of micrococcus luteus in indicator bacteria, sodium hydrogen phosphate 5.3g, dusty yeast 5g, glucose 5g, sodium chloride 5g, agar powder 12g, water 1000ml) flat board.Hatch 16～20h for 30 ℃, measure antibacterial circle diameter.To the mapping of tylosin concentration, can obtain regression equation and R by antibacterial circle diameter
2Value.
2) get tylosin herb residue sample 1.00g after the processing, add 5ml 0.01mol/L tartaric acid solution, after the stirring, soak 5～8h, after stirring again, leave standstill 10～30min.Get the 1ml leachate, the centrifugal 10min of 5000r/min.Getting supernatant 10 μ l point samples is the S 1 solid medium flat board of micrococcus luteus in indicator bacteria.Hatch 16～20h for 30 ℃, measure antibacterial circle diameter.According to the regression equation of step (1), obtain handling residual tylosin content in the dregs of a decoction of back.
The degradation bacteria strains of tylosin herb residue through filtering out handled, and handles the existence that does not detect residual tylosin in the dregs of a decoction of back.
The present invention has following effect:
1) method uniqueness: the present invention is by screening, cultivate the compound bacteria of residual tylosin in the dregs of a decoction of can degrading, and then from compound bacteria, separate, bacterial strain that purifying obtains degrading tylosin, prepare corresponding dregs of a decoction culture medium, insert residual tylosin in the degradation bacteria fermentative degradation dregs of a decoction.
2) the residual tylosin in the dregs of a decoction of effectively having degraded: the present invention adopts microbial method that tylosin herb residue is carried out degradation treatment, residual tylosin in the dregs of a decoction of can degrading fully.
3) the present invention can make tylosin herb residue utilize again becomes possibility, realizes the tylosin herb residue zero-emission.Because the residual quantity of tylosin is higher in the dregs of a decoction, tylosin herb residue can not directly be used as animal feed or fertilizer.The undressed tylosin herb residue if animal is fed for a long time, in the meat that produces, egg, the milk tylosin residual quantity up to 0.5mg/kg about, and food and drug administration (FDA) regulation, the residual safe level of tylosin in edible tissue or egg be not for being higher than 0.2mg/kg.If tylosin herb residue is directly with fertilizer form fertilising, residual tylosin may cause the increase of resistance salmonella quantity in the environment.
4) the present invention handles the dregs of a decoction by the tylosin degradation bacteria, and the residual tylosin in the dregs of a decoction of not only having degraded has also improved Protein content in the dregs of a decoction simultaneously.
The specific embodiment
Example 1:
The method of residual tylosin in a kind of dregs of a decoction of degrading, this method comprises the steps:
1) preparation of soil bacteria suspension: take by weighing and stack near the soil sample 1g tylosin herb residue, place triangular flask, add the 100ml PBS, after shaking up, place the constant temperature shaking table, under 30 ℃, the 200r/min 20min that vibrates;
2) strain improvement culture medium preparation: with the raw material of following weight or volume mix get final product the strain improvement culture medium: tylosin herb residue 200g, peptone 10g, glucose 10g, K
2HPO
40.1g, FeSO
47H
2O 0.2g, water 800ml;
3) strain domestication culture medium preparation: with following raw material mix by weight get final product the strain domestication culture medium: 1. one-level domestication culture medium: tylosin herb residue and water are prepared in 1: 9 ratio; 2. secondary is tamed culture medium: tylosin herb residue and water are prepared in 2: 8 ratio; 3. tame culture mediums for three grades: tylosin herb residue and water are prepared in 3: 7 ratio; 4. level Four is tamed culture medium: tylosin herb residue and water are prepared in 4: 6 ratio;
4) bacterial screening culture medium preparation: with the raw material of following weight or volume mix get final product the bacterial screening culture medium: 1. primary dcreening operation isolation medium: tylosin herb residue 200g, agar 20g, water 800ml; 2. multiple screening the: yeast extract powder 10g, peptone 20g, glucose 20g, Tylosin Tartrate standard items 0.04g, water 1000ml from culture medium; 3. separation and purification culture medium: peptone 10g, glucose 5g, tylosin herb residue leachate 1000ml;
5) actication of culture rejuvenation culture medium preparation: with the raw material of following weight or volume mix get final product actication of culture rejuvenation culture medium: yeast extract powder 10g, peptone 10g, tylosin herb residue leachate 1000ml;
6) preparation of dregs of a decoction fermentation medium: tylosin herb residue 700g, wheat bran 300g, K
2HPO
40.2g, FeSO
47H
2O 0.1g, water 900ml.
7) get above-mentioned steps 1) the soil bacteria suspension 20ml of preparation, join 100g above-mentioned steps 2) in the strain improvement culture medium of preparation, stir, putting in 30 ℃ of constant temperature shaking tables, 120r/min cultivates down 72h; Get the bacteria suspension after the cultivation, the inoculum concentration by 5% is linked into above-mentioned steps 3) in the first class inoculum domestication culture medium of preparation, put in 30 ℃ of constant temperature shaking tables, 120r/min cultivates 72h.Repeat this step 10 time, used strain domestication culture medium rises to level Four step by step by one-level;
8) getting nutrient solution 3ml after the domestication, is 10 with the PBS gradient dilution
-1, 10
-210
-10Concentration, adopt pour plate method will dilute back bacterium liquid then and be inoculated in the primary dcreening operation isolation medium, pour into a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices after the mixing, difference according to colonial morphology and growth cycle thereof, number consecutively also inserts liquid with it and sieves culture medium again, after cultivation is gone down to posterity, adopt the spread plate method to be inoculated into solid again and sieve culture medium again, through after the separation it being inserted fluid separation applications purifying culture medium once more, cultivation is gone down to posterity the back streak inoculation to solid separation and purification culture medium, and after repeating this step 4 time, can filter out the bacterial strain of degraded tylosin;
9) get above-mentioned steps 8) the tylosin degradation bacteria of gained, be linked in the actication of culture rejuvenation culture medium, under 30 ℃, cultivate 24h in the 120r/min constant temperature shaking table, behind the centrifugal 15min of 4000r/min, be suspended in the new actication of culture rejuvenation culture medium, 120r/min continue to cultivate 10h in 30 ℃ of constant temperature shaking tables, the bacteria suspension of the tylosin that can obtain degrading.
10) get the degraded tylosin bacteria suspension 2ml, be linked in the 100g dregs of a decoction fermentation medium, stir.Under 30 ℃, hatch 96h.80 ℃ of oven dry down, get final product after the pulverizing then.
Detect through microbial method, handle the existence that does not detect residual tylosin in the dregs of a decoction of back.
Example 2:
The method of residual tylosin in a kind of dregs of a decoction of degrading, this method comprises the steps:
1) preparation of soil bacteria suspension: take by weighing and stack near the soil sample 10g tylosin herb residue, place triangular flask, add the 200ml PBS, after shaking up, place the constant temperature shaking table, under 28 ℃, the 200r/min 40min that vibrates;
2) strain improvement culture medium preparation: with the raw material of following weight or volume mix get final product the strain improvement culture medium: tylosin herb residue 200g, peptone 10g, glucose 10g, K
2HPO
40.1g, FeSO
47H
2O 0.2g, water 800ml;
3) strain domestication culture medium preparation: with following raw material mix by weight get final product the strain domestication culture medium: 1. one-level domestication culture medium: tylosin herb residue and water are prepared in 1: 9 ratio; 2. secondary is tamed culture medium: tylosin herb residue and water are prepared in 2: 8 ratio; 3. tame culture mediums for three grades: tylosin herb residue and water are prepared in 3: 7 ratio; 4. level Four is tamed culture medium: tylosin herb residue and water are prepared in 4: 6 ratio;
4) bacterial screening culture medium preparation: with the raw material of following weight or volume mix get final product the bacterial screening culture medium: 1. primary dcreening operation isolation medium: tylosin herb residue 200g, agar 20g, water 800ml; 2. multiple screening the: yeast extract powder 10g, peptone 20g, glucose 20g, Tylosin Tartrate standard items 0.04g, water 1000ml from culture medium; 3. separation and purification culture medium: peptone 10g, glucose 5g, tylosin herb residue leachate 1000ml;
5) actication of culture rejuvenation culture medium preparation: with the raw material of following weight or volume mix get final product actication of culture rejuvenation culture medium: yeast extract powder 10g, peptone 10g, tylosin herb residue leachate 1000ml;
6) preparation of dregs of a decoction fermentation medium: tylosin herb residue 800g, wheat bran 200g, K
2HPO
40.15g, FeSO
47H
2O 0.15g, water 850ml;
7) get above-mentioned steps 1) the soil bacteria suspension 10ml of preparation, join 200g above-mentioned steps 2) in the strain improvement culture medium of preparation, stir, putting in 28 ℃ of constant temperature shaking tables, 120r/min cultivates down 72h; Get the bacteria suspension after the cultivation, the inoculum concentration by 2% is linked into above-mentioned steps 3) in the first class inoculum domestication culture medium of preparation, put in 28 ℃ of constant temperature shaking tables, 120r/min cultivates 72h.Repeat this step 8 time, used strain domestication culture medium rises to level Four step by step by one-level;
8) getting nutrient solution 1ml after the domestication, is 10 with the PBS gradient dilution
-1, 10
-210
-10Concentration, adopt pour plate method will dilute back bacterium liquid then and be inoculated in the primary dcreening operation isolation medium, pour into a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices after the mixing, difference according to colonial morphology and growth cycle thereof, number consecutively also inserts liquid with it and sieves culture medium again, after cultivation is gone down to posterity, adopt the spread plate method to be inoculated into solid again and sieve culture medium again, through after the separation it being inserted fluid separation applications purifying culture medium once more, cultivation is gone down to posterity the back streak inoculation to solid separation and purification culture medium, and after repeating this step 3 time, can filter out the bacterial strain of degraded tylosin;
9) get above-mentioned steps 8) degraded tylosin bacterial strain, be linked into above-mentioned steps 5) preparation actication of culture rejuvenation culture medium in, .28 ℃ under, cultivate 26h in the 120r/min constant temperature shaking table, behind the centrifugal 15min of 4000r/min, be suspended in the new actication of culture rejuvenation culture medium, 120r/min continue to cultivate 10h in 28 ℃ of constant temperature shaking tables, the bacteria suspension of the tylosin that can obtain degrading.
10) get the degraded tylosin bacteria suspension 10ml, be linked in the 200g dregs of a decoction fermentation medium, stir.Under 28 ℃, hatch 108h.80 ℃ of oven dry down, get final product after the pulverizing then.
Detect through microbial method, handle the existence that does not detect residual tylosin in the dregs of a decoction of back.
Example 3:
The method of residual tylosin in a kind of dregs of a decoction of degrading, this method comprises the steps:
1) preparation of soil bacteria suspension: take by weighing and stack near the soil sample 5g tylosin herb residue, place triangular flask, add the 150ml PBS, after shaking up, place the constant temperature shaking table, under 35 ℃, the 200r/min 30min that vibrates;
2) strain improvement culture medium preparation: with the raw material of following weight or volume mix get final product the strain improvement culture medium: tylosin herb residue 200g, peptone 10g, glucose 10g, K
2HPO
40.1g, FeSO
47H
2O 0.2g, water 800ml;
3) strain domestication culture medium preparation: with following raw material mix by weight get final product the strain domestication culture medium: 1. one-level domestication culture medium: tylosin herb residue and water are prepared in 1: 9 ratio; 2. secondary is tamed culture medium: tylosin herb residue and water are prepared in 2: 8 ratio; 3. tame culture mediums for three grades: tylosin herb residue and water are prepared in 3: 7 ratio; 4. level Four is tamed culture medium: tylosin herb residue and water are prepared in 4: 6 ratio;
4) bacterial screening culture medium preparation: with the raw material of following weight or volume mix get final product the bacterial screening culture medium: 1. primary dcreening operation isolation medium: tylosin herb residue 200g, agar 20g, water 800ml; 2. multiple screening the: yeast extract powder 10g, peptone 20g, glucose 20g, Tylosin Tartrate standard items 0.04g, water 1000ml from culture medium; 3. separation and purification culture medium: peptone 10g, glucose 5g, tylosin herb residue leachate 1000ml;
5) actication of culture rejuvenation culture medium preparation: with the raw material of following weight or volume mix get final product actication of culture rejuvenation culture medium: yeast extract powder 10g, peptone 10g, tylosin herb residue leachate 1000ml;
6) preparation of dregs of a decoction fermentation medium: tylosin herb residue 900g, wheat bran 100g, K
2HPO
40.1g, FeSO
47H
2O 0.2g, water 800ml;
7) get above-mentioned steps 1) the soil bacteria suspension 10ml of preparation, join 150g above-mentioned steps 2) in the strain improvement culture medium of preparation, stir, putting in 35 ℃ of constant temperature shaking tables, 120r/min cultivates down 60h; Get the bacteria suspension after the cultivation, the inoculum concentration by 3% is linked into above-mentioned steps 3) in the first class inoculum domestication culture medium of preparation, put in 35 ℃ of constant temperature shaking tables, 120r/min cultivates 60h.Repeat this step 5 time, used strain domestication culture medium rises to level Four step by step by one-level;
8) getting nutrient solution 2ml after the domestication, is 10 with the PBS gradient dilution
-1, 10
-210
-10Concentration, adopt pour plate method will dilute back bacterium liquid then and be inoculated in the primary dcreening operation isolation medium, pour into a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices after the mixing, difference according to colonial morphology and growth cycle thereof, number consecutively also inserts liquid with it and sieves culture medium again, after cultivation is gone down to posterity, adopt the spread plate method to be inoculated into solid again and sieve culture medium again, through after the separation it being inserted fluid separation applications purifying culture medium once more, cultivation is gone down to posterity the back streak inoculation to solid separation and purification culture medium, and after repeating this step 3 time, can filter out the bacterial strain of degraded tylosin;
9) get above-mentioned steps 8) degraded tylosin bacterial strain, be linked into above-mentioned steps 5) preparation actication of culture rejuvenation culture medium in, under 35 ℃, cultivate 20h in the 120r/min constant temperature shaking table, behind the centrifugal 15min of 4000r/min, be suspended in the new actication of culture rejuvenation culture medium, 120r/min continue to cultivate 8h in 35 ℃ of constant temperature shaking tables, the bacteria suspension of the tylosin that can obtain degrading.
10) get the degraded tylosin bacteria suspension 8ml, be linked in the 150g dregs of a decoction fermentation medium, stir.Under 35 ℃, hatch 60h.100 ℃ of oven dry down, get final product after the pulverizing then.
Detect through microbial method, handle the existence that does not detect residual tylosin in the dregs of a decoction of back.
Example 4:
The method of residual tylosin in a kind of dregs of a decoction of degrading, this method comprises the steps:
1) preparation of soil bacteria suspension: take by weighing and stack near the soil sample 8g tylosin herb residue, place triangular flask, add the 200ml PBS, after shaking up, place the constant temperature shaking table, under 25 ℃, the 200r/min 40min that vibrates;
2) strain improvement culture medium preparation: with the raw material of following weight or volume mix get final product the strain improvement culture medium: tylosin herb residue 200g, peptone 10g, glucose 10g, K
2HPO
40.1g, FeSO
47H
2O 0.2g, water 800ml;
3) strain domestication culture medium preparation: with following raw material mix by weight get final product the strain domestication culture medium: 1. one-level domestication culture medium: tylosin herb residue and water are prepared in 1: 9 ratio; 2. secondary is tamed culture medium: tylosin herb residue and water are prepared in 2: 8 ratio; 3. tame culture mediums for three grades: tylosin herb residue and water are prepared in 3: 7 ratio; 4. level Four is tamed culture medium: tylosin herb residue and water are prepared in 4: 6 ratio;
4) bacterial screening culture medium preparation: with the raw material of following weight or volume mix get final product the bacterial screening culture medium: 1. primary dcreening operation isolation medium: tylosin herb residue 200g, agar 20g, water 800ml; 2. multiple screening the: yeast extract powder 10g, peptone 20g, glucose 20g, Tylosin Tartrate standard items 0.04g, water 1000ml from culture medium; 3. separation and purification culture medium: peptone 10g, glucose 5g, tylosin herb residue leachate 1000ml;
5) actication of culture rejuvenation culture medium preparation: with the raw material of following weight or volume mix get final product actication of culture rejuvenation culture medium: yeast extract powder 10g, peptone 10g, tylosin herb residue leachate 1000ml;
6) preparation of dregs of a decoction fermentation medium: tylosin herb residue 800g, wheat bran 200g, K
2HPO
40.2g, FeSO
47H
2O 0.1g, water 800ml;
7) get above-mentioned steps 1) the soil bacteria suspension 15ml of preparation, join 200g above-mentioned steps 2) in the strain improvement culture medium of preparation, stir, putting in 25 ℃ of constant temperature shaking tables, 120r/min cultivates down 72h; Get the bacteria suspension after the cultivation, the inoculum concentration by 4% is linked into above-mentioned steps 3) in the first class inoculum domestication culture medium of preparation, put in 25 ℃ of constant temperature shaking tables, 120r/min cultivates 72h.Repeat this step 8 time, used strain domestication culture medium rises to level Four step by step by one-level;
8) getting nutrient solution 2ml after the domestication, is 10 with the PBS gradient dilution
-1, 10
-210
-10Concentration, adopt pour plate method will dilute back bacterium liquid then and be inoculated in the primary dcreening operation isolation medium, pour into a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices after the mixing, difference according to colonial morphology and growth cycle thereof, number consecutively also inserts liquid with it and sieves culture medium again, after cultivation is gone down to posterity, adopt the spread plate method to be inoculated into solid again and sieve culture medium again, through after the separation it being inserted fluid separation applications purifying culture medium once more, cultivation is gone down to posterity the back streak inoculation to solid separation and purification culture medium, and after repeating this step 4 time, can filter out the bacterial strain of degraded tylosin;
9) get above-mentioned steps 8) degraded tylosin bacterial strain, be linked into above-mentioned steps 5) preparation actication of culture rejuvenation culture medium in, under 25 ℃, cultivate 30h in the 120r/min constant temperature shaking table, behind the centrifugal 15min of 4000r/min, be suspended in the new actication of culture rejuvenation culture medium, 120r/min continue to cultivate 14h in 25 ℃ of constant temperature shaking tables, the bacteria suspension of the tylosin that can obtain degrading.
10) get the degraded tylosin bacteria suspension 10ml, be linked in the 100g dregs of a decoction fermentation medium, stir.Under 25 ℃, hatch 120h.60 ℃ of oven dry down, get final product after the pulverizing then.
Detect through microbial method, handle the existence that does not detect residual tylosin in the dregs of a decoction of back.
Example 5:
The method of residual tylosin in a kind of dregs of a decoction of degrading, this method comprises the steps:
1) preparation of soil bacteria suspension: take by weighing and stack near the soil sample 3g tylosin herb residue, place triangular flask, add the 100ml PBS, after shaking up, place the constant temperature shaking table, under 32 ℃, the 200r/min 25min that vibrates;
2) strain improvement culture medium preparation: with the raw material of following weight or volume mix get final product the strain improvement culture medium: tylosin herb residue 200g, peptone 10g, glucose 10g, K
2HPO
40.1g, FeSO
47H
2O 0.2g, water 800ml;
3) strain domestication culture medium preparation: with following raw material mix by weight get final product the strain domestication culture medium: 1. one-level domestication culture medium: tylosin herb residue and water are prepared in 1: 9 ratio; 2. secondary is tamed culture medium: tylosin herb residue and water are prepared in 2: 8 ratio; 3. tame culture mediums for three grades: tylosin herb residue and water are prepared in 3: 7 ratio; 4. level Four is tamed culture medium: tylosin herb residue and water are prepared in 4: 6 ratio;
4) bacterial screening culture medium preparation: with the raw material of following weight or volume mix get final product the bacterial screening culture medium: 1. primary dcreening operation isolation medium: tylosin herb residue 200g, agar 20g, water 800ml; 2. multiple screening the: yeast extract powder 10g, peptone 20g, glucose 20g, Tylosin Tartrate standard items 0.04g, water 1000ml from culture medium; 3. separation and purification culture medium: peptone 10g, glucose 5g, tylosin herb residue leachate 1000ml;
5) actication of culture rejuvenation culture medium preparation: with the raw material of following weight or volume mix get final product actication of culture rejuvenation culture medium: yeast extract powder 10g, peptone 10g, tylosin herb residue leachate 1000ml;
6) preparation of dregs of a decoction fermentation medium: tylosin herb residue 700g, wheat bran 300g, K
2HPO
40.15g, FeSO
47H
2O 0.15g, water 850ml;
7) get above-mentioned steps 1) the soil bacteria suspension 15ml of preparation, join 100g above-mentioned steps 2) in the strain improvement culture medium of preparation, stir, putting in 32 ℃ of constant temperature shaking tables, 120r/min cultivates down 60h; Get the bacteria suspension after the cultivation, the inoculum concentration by 4% is linked into above-mentioned steps 3) in the first class inoculum domestication culture medium of preparation, put in 32 ℃ of constant temperature shaking tables, 120r/min cultivates 60h.Repeat this step 8 time, used strain domestication culture medium rises to level Four step by step by one-level;
8) getting nutrient solution 3ml after the domestication, is 10 with the PBS gradient dilution
-1, 10
-210
-10Concentration, adopt pour plate method will dilute back bacterium liquid then and be inoculated in the primary dcreening operation isolation medium, pour into a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices after the mixing, difference according to colonial morphology and growth cycle thereof, number consecutively also inserts liquid with it and sieves culture medium again, after cultivation is gone down to posterity, adopt the spread plate method to be inoculated into solid again and sieve culture medium again, through after the separation it being inserted fluid separation applications purifying culture medium once more, cultivation is gone down to posterity the back streak inoculation to solid separation and purification culture medium, and after repeating this step 4 time, can filter out the bacterial strain of degraded tylosin;
9) get above-mentioned steps 8) degraded tylosin bacterial strain, be linked into above-mentioned steps 5) preparation actication of culture rejuvenation culture medium in, under 32 ℃, cultivate 24h in the 120r/min constant temperature shaking table, behind the centrifugal 15min of 4000r/min, be suspended in the new actication of culture rejuvenation culture medium, 120r/min continue to cultivate 10h in 32 ℃ of constant temperature shaking tables, the bacteria suspension of the tylosin that can obtain degrading.
10) get the degraded tylosin bacteria suspension 7ml, be linked in the 100g dregs of a decoction fermentation medium, stir.Under 32 ℃, hatch 72h.100 ℃ of oven dry down, get final product after the pulverizing then.
Detect through microbial method, handle the existence that does not detect residual tylosin in the dregs of a decoction of back.
Above-mentioned steps 4) tylosin herb residue leachate is that the ratio according to dregs of a decoction weight in wet base and water is preparation in 1: 2, and concrete steps are as follows:
A. get tylosin herb residue 100g, add sterilized distilled water of 200ml or deionized water, at room temperature soak 12～20h after stirring;
B. soak among the above-mentioned steps a is sub-packed in the centrifuge tube, gradient centrifugation 30min abandons precipitation;
C. get the supernatant after the gradient centrifugation, abandon floating impurity by suction filtration again and get final product;
Above-mentioned steps 7) the domestication process of tylosin degradation bacteria has experienced two stages, and it is followed successively by stabilization sub stage of bacterial strain proterties and the bacterial strain stabilization sub stage to the tylosin degradation capability.
Claims (3)
1. the method for residual tylosin in the dregs of a decoction of degrading, it is characterized in that: this method comprises the steps:
1) preparation of soil bacteria suspension: use quartering, take by weighing and stack near the soil sample 1～10g of tylosin herb residue, place triangular flask, add 100～200ml PBS, after shaking up, place the constant temperature shaking table, under 25～35 ℃, the 200r/min 20～40min that vibrates;
2) strain improvement culture medium preparation: with the raw material of following weight mix get final product the strain improvement culture medium: tylosin herb residue 200g, peptone 10g, glucose 10g, K
2HPO
40.1g, FeSO
47H
2O 0.2g, water 800ml;
3) strain domestication culture medium preparation: with following raw material mix by weight get final product the strain domestication culture medium: 1. one-level domestication culture medium: tylosin herb residue and water are prepared in 1: 9 ratio; 2. secondary is tamed culture medium: tylosin herb residue and water are prepared in 2: 8 ratio; 3. tame culture mediums for three grades: tylosin herb residue and water are prepared in 3: 7 ratio; 4. level Four is tamed culture medium: tylosin herb residue and water are prepared in 4: 6 ratio;
4) bacterial screening culture medium preparation: with the raw material of following weight mix get final product the bacterial screening culture medium: 1. primary dcreening operation isolation medium: tylosin herb residue 200g, agar 20g, water 800ml; 2. multiple screening the: yeast extract powder 10g, peptone 20g, glucose 20g, Tylosin Tartrate standard items 0.04g, water 1000ml from culture medium; 3. separation and purification culture medium: peptone 10g, glucose 5g, tylosin herb residue leachate 1000ml;
5) actication of culture rejuvenation culture medium preparation: with the raw material of following weight mix get final product actication of culture rejuvenation culture medium: yeast extract powder 10g, peptone 10g, tylosin herb residue leachate 1000ml;
6) preparation of dregs of a decoction fermentation medium: with the raw material of following weight mix get final product dregs of a decoction fermentation medium: tylosin herb residue 700～900g, wheat bran 300～100g, K
2HPO
40.1～0.2g, FeSO
47H
2O 0.1～0.2g, water 800～900ml;
7) get above-mentioned steps 1) the soil bacteria suspension 10～20ml of preparation, join 100～200g above-mentioned steps 2) in the strain improvement culture medium of preparation, stir, putting in 25～35 ℃ of constant temperature shaking tables, 120r/min cultivates down 60～72h; Get the bacteria suspension after the cultivation, inoculum concentration by 2～5% is linked into above-mentioned steps 3) in the first class inoculum domestication culture medium of preparation, put in 25～35 ℃ of constant temperature shaking tables, 120r/min cultivates 60～72h, repeat this step 5～10 time, used strain domestication culture medium rises to level Four step by step by one-level;
8) getting nutrient solution 1～3ml after the domestication, is 10 with the PBS gradient dilution
-1, 10
-2... 10
-10Concentration, adopt pour plate method will dilute back bacterium liquid then and be inoculated in the primary dcreening operation isolation medium, pour into a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices after the mixing, difference according to colonial morphology and growth cycle thereof, number consecutively also inserts liquid with it and sieves again from culture medium, adopting the spread plate method to be inoculated into solid after cultivation is gone down to posterity again sieves again from culture medium, through after the separation it being inserted fluid separation applications purifying culture medium once more, cultivation is gone down to posterity the back streak inoculation to solid separation and purification culture medium, and after repeating this step 3～4 time, finish the separation and purification of bacterial classification, can filter out the bacterial strain of degraded tylosin;
9) get above-mentioned steps 8) degraded tylosin bacterial strain, be linked into above-mentioned steps 5) preparation actication of culture rejuvenation culture medium in, put in 25～35 ℃ of constant temperature shaking tables, 120r/min cultivates 20～30h down, behind the centrifugal 15min of 4000r/min, be suspended in the new actication of culture rejuvenation culture medium, 120r/min continues to cultivate 8～14h in 25～35 ℃ of constant temperature shaking tables, can obtain tylosin degradation bacteria suspension;
10) get above-mentioned steps 6) the dregs of a decoction fermentation medium 100～200g of preparation, insert above-mentioned steps 9) the tylosin degradation bacteria suspension 2～10ml of preparation, and stirring, 25～35 ℃ of fermentation 60～120h 60～100 ℃ of oven dry down, get final product after the pulverizing then.
2. the method for residual tylosin in a kind of dregs of a decoction of degrading as claimed in claim 1 is characterized in that: the tylosin herb residue leachate of described step 4) is that the ratio according to dregs of a decoction weight in wet base and water is preparation in 1: 2, and concrete steps are as follows:
A. get tylosin herb residue 100g, add sterilized distilled water of 200ml or deionized water, at room temperature soak 12～20h after stirring;
B. soak among the above-mentioned steps a is sub-packed in the centrifuge tube, gradient centrifugation 30min abandons precipitation;
C. get the supernatant after the gradient centrifugation, abandon floating impurity by suction filtration again and get final product.
3. the method for residual tylosin in a kind of dregs of a decoction of degrading as claimed in claim 1, it is characterized in that: the domestication process of described step 7) tylosin degradation bacteria has experienced two stages, and it is followed successively by stabilization sub stage of bacterial strain proterties and the bacterial strain stabilization sub stage to the tylosin degradation capability.
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| CN1778316A (en) * | 2004-11-26 | 2006-05-31 | 广东省新的生物工程研究所有限公司广州生物制品厂 | Degradation of bacterial drug resistance by composite microbial preparation |
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