CN103931965A - Method used for biodegradation of patulin with Rhodosporidium paludigenum Fell&Tallman - Google Patents
Method used for biodegradation of patulin with Rhodosporidium paludigenum Fell&Tallman Download PDFInfo
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- CN103931965A CN103931965A CN201410058749.1A CN201410058749A CN103931965A CN 103931965 A CN103931965 A CN 103931965A CN 201410058749 A CN201410058749 A CN 201410058749A CN 103931965 A CN103931965 A CN 103931965A
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Abstract
The invention discloses a method used for biodegradation of patulin with Rhodosporidium paludigenum Fell&Tallman. According to the method, a Rhodosporidium paludigenum Fell&Tallman bacterial suspension is added into a liquid material contaminated by patulin until a final concentration of Rhodosporidium paludigenum Fell&Tallman reaches 90000 to 110000cfu/ml; an obtained mixture is subjected to degradation processing for 2 to 4 days at a temperature of 24 to 26 DEG C in a shaking table with a shaking speed of 150 to 250rpm so as to realize degradation of patulin. The method is used for degrading patulin, is low in cost, is excellent in degradation effects, and is high in safety; and no secondary pollution is caused.
Description
Technical field
The invention belongs to microbial bacterial agent field, particularly, relate to a kind of method of utilizing safe marine yeast degradative fungi toxin clavacin.
Background technology
Clavacin (patulin, 4-hydroxy-4H-furo[3,2-c] pyran-2 (6H)-one) be a kind of water miscible macrolide, be present in some rotten fruits (as apple, oranges and tangerines, pomegranate, pears etc.) and goods thereof.Clavacin can be produced by many fungies, mainly comprise aspergillus fungi (Aspergillus), Penicillium fungi (Penicillium) and paecilomyces fungi (Paecilomyces), wherein Penicillium fungi is the main producer of occurring in nature clavacin.
Clavacin is a kind of to the virose mycotoxin of people and animals.In report, known acute toxicity comprises nauseating, ulcer at present; Chronic toxicity comprises neurotoxicity, immunotoxicity, genotoxicity, Immunosuppression system, carcinogenic teratogenesis mutagenesis etc.In addition, in molecular biology aspect, clavacin can suppress that DNA is synthetic, protein crosslinked, react etc. with glutathione.Clavacin in the apple goods that various countries scientist produces this country and citrus goods (being mainly cider) detects, find Some European country, Korea S and Japan, control to clavacin in food is better, and some are local as Spain, Italy, South Africa, India and China etc., in food, the recall rate of clavacin is higher, and sample segment exceeds standard very serious.Such as researcher extracts the detection that apple goods carry out clavacin recently from Manchurian supermarket, find that 16% product clavacin content overproof (reaches as high as 97.7ppb, and GB regulation is lower than 50ppb), only have its clavacin content of sample of 12.6% lower than detectability.
Clavacin is the secondary metabolic product that penicillium expansum produces.Although people have had a lot of fruit and vegetable fresh-keeping agents, bactericide and fresh-keeping means (as low temperature accumulating, controlled atmosphere etc.) now, the secretion of Antifungi growth and reduction mycotoxin might not be correlated with between the two.Existing many materials for preserving fruit and vegetable utilizing or means (as low temperature, controlled atmosphere etc.) are although can effectively reduce the generation of diseases of garden stuff, but they to the control of corresponding mycotoxin also to no effect, on the contrary, there are some fresh products or fresh-keeping means to impel on the contrary fungi to produce more toxin.This is because many fungies, in being unfavorable for the adverse circumstance of growth, can secrete the secondary metabolic products such as more mycotoxin, strengthens the competitiveness of self.Example, it is reported, in experiment in vitro, while adding the bactericide such as captan (Captan), carbendazim (Carbendazim), bupirimate (Bupirimate) in the culture medium of cultivating penicillium expansum, can induce penicillium expansum to produce more clavacin.Separately there are some researches show, the clavacin content in organic apple of processing through biologic product, higher than the apple through traditional agriculture (employing bactericide) cultivation, does not also form final conclusion to the explanation of this phenomenon.Seminar is studied the apple of ca cold storage, although there were significant differences on the incidence of disease for the apple that discovery is processed through different controlled atmospheres, but indifference on the content of clavacin, illustrates herein under reason, the size of scab and the content of clavacin are also uncorrelated.Generally speaking, although antistaling agent and fresh-keeping means can be controlled the growth of fungi, can not reduce the amount of fungus secretion clavacin.
Because clavacin is very stable under sour environment, once therefore there is (fruit and the goods thereof that are polluted by clavacin mostly are sour environment), be just difficult to remove by conventional method.There is at present report to utilize the physico-chemical processes such as ozone, radioactive element, heat treatment, macroporous resin adsorption, clavacin is removed.But the cost that these methods have is higher, what have has negative effect to environment or human body, and some degradation effects are not good enough, therefore also not bery desirable.Comparatively speaking, biological method is safer, low-cost.Many bibliographical informations show, utilize microorganism to degrade to clavacin, not only very effectively, and with low cost, safe, the toxicity of its catabolite is also little than clavacin itself.The microorganism of the degradable clavacin of having reported at present comprises micropopulation, rhodotorula (Rhodosporidium kratochvilovae) and the Pichia pastoris (Pichia membranifaciens and Pichia caribbica) etc. in oxidizing glucose acidfast bacilli (Gluconobacter oxydans), saccharomyces cerevisiae (Saccharomyces cerevisiae, need under oxygen-free environment), cud.
Red winter spore yeast (Rhodosporidium paludigenum Fell & Tallman) is the biological and ecological methods to prevent plant disease, pests, and erosion yeast of the safety separated from ocean of a strain.According to the study, this yeast can adapt to the environment of hyperosmosis, and controls very effective to the postharvest disease of little tomato, winter jujube and oranges and tangerines.But not there are some researches show at present, red winter spore has degradation to clavacin itself.
This red winter spore yeast (Rhodosporidium paludigenum Fell & Tallman) also this marine yeast of name (Rhodosporidium paludigenum Fell & Tallman) is preserved in Britain international standard fungal studies institute's International Agriculture and Bio-Centers genetic resources preservation center (International Mycological Institute, CABI Genetic Resource Collection), preservation address: Britain TW209TY Surrey Jun Aige town Beckham road International Agriculture and Bio-Centers Britain center, preservation date: 2006.4.19, deposit number: IMI394084.
Above-mentioned marine yeast cell suspending liquid has been used to produce bio-preservative, and the application number of this patent is 200610155209.0.
The list of references above relating to is specific as follows:
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Cooray R, Kiessling KH, Lindahl-Kiessling K (1982) .The effects of patulin and patulin-cysteine mixtures on DNA synthesis and the frequency of sister-chromatid exchanges in human lymphocytes.Food and Chemical Toxicology, 20 (6), 893-898(Cooray R, Kiessling KH, Lindahl-Kiessling K (1982). the impact that clavacin and clavacin-cysteine compound are synthetic on the DNA of mankind's lymphocytes and sisters monosome exchanges.Food and chemical substance toxicology.20(6),893-898）；
G.Wichmann, O.Herbarth, I.Lehmann (2002) .The mycotoxins citrinin, gliotoxin, and patulin affect interferon-γ rather than interleukin-4production in human blood cells.Environmental Toxicology, 17 (3), 211-218(G.Wichmann, O.Herbarth, I.Lehmann (2002). mycotoxin notalin, trichodermin and clavacin are greater than the synthetic of interleukin-4 to the synthetic impact of interferon-γ in mankind's haemocyte.Ecotoxicology.17(3),211-218）；
Glaser, N.and H.Stopper (2012) .Patulin:Mechanism of genotoxicity.Food and Chemical Toxicology50 (5): 1796-1801(Glaser, N.and H.Stopper (2012). clavacin: the mechanism of genotoxicity.Food and chemical substance toxicology.50(5):1796-1801）；
K Walker, BP Wiesner (1944) .Patulin and clavicin.Lancet, 246:294(K Walker, BP Wiesner (1944). clavacin, lancet, 246:294);
L Escoula, J More, C Baradat-Annales de Recherches Veterinaires (1977) .The toxins of Byssochlamys nivea westling.-Acute toxicity of patulin in adult rats and mice.Annals of veterinary research, 8 (1): 41-49(L Escoula, J More, the toxin of C Baradat-Annales de Recherches Veterinaires (1977) .Byssochlamys nivea westling: the acute toxicity of clavacin to adult rat and mouse.Animal doctor studies annual.8(1):41-49）；
Morales, H., V.Sanchis, et al. (2007) .Patulin accumulation in apples during postharvest:Effect of controlled atmosphere storage and fungicide treatments.Food Control, 18 (11): 1443-1448(Morales, H., V.Sanchis, et al. (2007). adopt the accumulation of clavacin in rear apple: the impact of ca cold storage and chemical sterilization machine.Food is controlled.18(11):1443-1448）；
Moake, M.M., Padilla-Zakour, O.I., Worobo, R.W. (2005) .Comprehensive review of patulin control methods in foods.Comprehensive Reviews in Food Science and Food Safety, 4:8-21(Moake, M.M., Padilla-Zakour, O.I., Worobo, R.W. (2005). control the method comprehensive review of clavacin in food.The comprehensive review of Food Science and food security.4:8-21）；
Morgavi, D.P., Boudra, H., Jouany, J.-P., Graviou, D. (2003) .Prevention of Patulin Toxicity on Rumen Microbial Fermentation by SH-Containing Reducing Agents.Journal of Agricultural and Food Chemistry, 51 (23): 6906-6910(Morgavi, D.P., Boudra, H., Jouany, J.-P., Graviou, D. (2003). the reproducibility chemical substance that comprises SH key reduces the toxic effect of clavacin to rumen microbial fermentation.Agriculture and food science.51(23):6906-6910）；
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Paterson, R.R.M. (2007) .Some fungicides and growth inhibitor/biocontrol-enhancer2-deoxy-d-glucose increase patulin from Penicillium expansum strains in vitro.Crop Protection26 (4): 543-548(Paterson, R.R.M. (2007). several chemical bactericides and GIF/biological and ecological methods to prevent plant disease, pests, and erosion control-intensive 2-deoxidation-d-glucose strengthen penicillium expansum secretion clavacin.Crop protection.26(4):543-548）；
Piamontese, L., Solfrizzo, M., & Visconti, A. (2005) .Occurrence of patulin in conventional and organic fruit products in Italy and subsequent exposure assessment.Food Additives and Contaminants, 22,437 – 442(Piamontese, L., Solfrizzo, M., & Visconti, A. (2005). in Italian tradition and organic fruits goods there is situation and risk assessment thereof in clavacin.Food additives and pollutant.22:437–442）；
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Summary of the invention
The technical problem to be solved in the present invention is to provide that a kind of cost is low, good degrading effect, security are good, the method for utilizing red winter spore yeast bio degraded clavacin of non-secondary pollution.
In order to solve the problems of the technologies described above, the invention provides a kind of method of utilizing red winter spore yeast bio degraded clavacin, the bacteria suspension of Rhodosporidium paludigenum Fell & Tallman is put into the liquid object of being polluted by clavacin, until the final concentration of Rhodosporidium paludigenum Fell & Tallman is 0.9～1.1 * 10
5cFU/mL(is for example 1 * 10
5cFU/mL), at 150～250rpm(, be preferably 180rpm) shaking table in 24～26 ℃ of (better 25 ℃) degradation treatment 2～4 days; Thereby realize the degraded of clavacin.
As the improvement that utilizes the method for red winter spore yeast bio degraded clavacin of the present invention:
The pH < 7(of the liquid object of being polluted by clavacin is preferably pH≤6).
As the further improvements in methods of utilizing red winter spore yeast bio degraded clavacin of the present invention:
The preparation method of the bacteria suspension of Rhodosporidium paludigenum Fell & Tallman is:
From NYDA medium slant, the colony inoculation of picking Rhodosporidium paludigenum Fell & Tallman, in NYDB nutrient solution, is preferably 180rpm in 24～26 ℃ of (better 25 ℃), 150～250rpm() condition under activate; Then be adjusted in the Rhodosporidium paludigenum Fell & Tallman bacteria suspension of every ml and contain 4～6 * 10
6cFU(is for example 5 * 10
6cFU) Rhodosporidium paludigenum Fell & Tallman thalline;
The formula of NYDA culture medium is: 10g glucose+8g beef extract+5g yeast extract powder+20g agar+1L distilled water;
The formula of NYDB nutrient solution is: 10g glucose+8g beef extract+5g yeast extract powder+10g sodium chloride+1L distilled water; Regulate pH value to 6.0.
As the further improvements in methods of utilizing red winter spore yeast bio degraded clavacin of the present invention:
Activation is included in the first generation in NYDB nutrient solution and cultivates 24h, and the second generation is cultivated 36h.
Remarks explanation: the formula of the NYDA culture medium in the present invention all belongs to routine techniques, and NYDB nutrient solution belongs to the formula after improvement.
NYDA:10g glucose+8g beef extract+5g yeast extract powder+20g agar+1L distilled water; Then high-temperature sterilization (be conventional high-temperature sterilization, be generally at 1.1 atmospheric pressure, 121 ℃ at sterilizing 20min).
NYDB:10g glucose+8g beef extract+5g yeast extract powder+10g sodium chloride+1L distilled water; Regulate pH value to 6.0(employing usual manner to regulate); Then high-temperature sterilization (be conventional high-temperature sterilization, be generally at 1.1 atmospheric pressure, 121 ℃ at sterilizing 20min).
Employing through two generation Liquid Culture red winter spore yeast, be the thalline that there is better activity in order to obtain.
The present invention is that to take the suspension of bacterial strain Rhodosporidium paludigenum Fell & Tallman be effective ingredient, puts it in the liquid object of being polluted by clavacin, thereby reaches the effect of biodegradation clavacin.
In the present invention, Rhodosporidium paludigenum Fell & Tallman, be red winter spore yeast, Classification And Nomenclature is Rhodosporidium paludigenum, strain number is Fell & Tallman, be preserved in Britain international standard fungal studies institute's International Agriculture and Bio-Centers genetic resources preservation center (International Mycological Institute, CABI Genetic Resource Collection), deposit number: IMI394084, refers to www.cabi.org.Clavacin, is mark product, purchased from life work bioengineering (Shanghai) limited company.
In the present invention, the method for counting by blood counting chamber, the amount of the thalline of control and regulation Rhodosporidium paludigenum Fell & Tallman bacteria suspension.
Research shows the Rhodosporidium paludigenum Fell & Tallman bacteria suspension clavacin of effectively degrading, and through further test, confirms, its dominant mechanism is biodegradation (enzyme reaction).Along with the great attention of people to food security, utilize the method for safe and effective Rhodosporidium paludigenum Fell & Tallman degraded clavacin to have wide actual application prospect and researching value.
Tool of the present invention has the following advantages:
1), degradation efficiency is high, effect is good:
The present invention adopts Rhodosporidium paludigenum Fell & Tallman biodegradation clavacin, the liquid object (for example fruit juice) that can effectively degrade and be polluted by clavacin within a short period of time, and degradation efficiency is high; And clavacin is degraded, the toxicity of afterproduct, well below the toxicity of clavacin itself, does not cause secondary pollution.Meanwhile, utilize the biological method of red winter spore yeast degradation clavacin compared with some physics, chemical method, there is the advantages such as safe, pollution-free, efficient.Therefore, method of the present invention can be promoted the use of on a large scale.
2), with low cost, easily obtain:
Rhodosporidium paludigenum Fell & Tallman is natural, the safe yeast strain obtaining from ocean separation, is present in the Nature, and wide material sources, are easy to obtain.Rhodosporidium paludigenum Fell & Tallman requires not harsh to growing environment, and osmophilic strain, is easy to cultivate, with low cost, therefore can cultivate in a large number, is applied to reality.
3), easy to use, simple to operate.
The Rhodosporidium paludigenum Fell & Tallman bacterial strain that the present invention adopts is easy to cultivate, and only needs by most basic microbial inoculant, cultivates operation, can obtain the thallus suspension liquid with high degrading activity.
Actual usage of the present invention and consumption are as follows:
At the contaminated liquid object of the clavacin (concentration≤200ppm of clavacin, be preferably the concentration≤30ppm of clavacin) in add the Rhodosporidium paludigenum Fell & Tallman thalline that activated to make bacteria suspension, until the ultimate density of thalline is 0.9～1.1 * 10
5cFU/mL(the best is 1 * 10
5cFU/mL).
Accompanying drawing explanation
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in further detail.
Fig. 1 is degrade in the NYDB design sketch of clavacin of Rhodosporidium paludigenum Fell & Tallman bacteria suspension of the present invention.
Fig. 2 is degrade in the cider effect figure of clavacin of Rhodosporidium paludigenum Fell & Tallman bacteria suspension of the present invention.
Fig. 3 is that clavacin is to colibacillary toxicity figure.
Fig. 4 is that Rhodosporidium paludigenum Fell & Tallman bacteria suspension of the present invention is degraded after clavacin in NYDB, and catabolite is to colibacillary toxicity figure.
The specific embodiment
Below by embodiment, the present invention is described in further detail.
Clavacin in embodiment 1, red winter spore yeast bio degraded NYDB, carries out following steps successively:
1), the preparation of bacteria suspension:
From NYDA medium slant, single colony inoculation of picking Rhodosporidium paludigenum Fell & Tallman is in the 250mL conical flask of NYDB that 50mL is housed, in 25 ℃, under the condition of 180rpm, activate, activation process is included in the first generation in NYDB and cultivates 24h, and the second generation is cultivated 36h; That is, in 25 ℃, the shaking table of 180rpm rotating speed, cultivate 24 hours; Then under aseptic condition, draw 1mL thallus suspension liquid in the 250mL conical flask of the NYDB that 50mL is housed of fresh sterilizing, in 25 ℃, the shaking table of 180rpm, cultivate 36 hours.
Then be adjusted in the Rhodosporidium paludigenum Fell & Tallman bacteria suspension of every ml and contain 5 * 10
6the Rhodosporidium paludigenum Fell & Tallman thalline of CFU.
NYDA:10g glucose+8g beef extract+5g yeast extract powder+20g agar+1L distilled water, then carries out conventional autoclaving.
NYDB:10g glucose+8g beef extract+5g yeast extract powder+10g sodium chloride+1L distilled water, regulates pH value to 6.0, then carries out conventional autoclaving.
2), the degraded of clavacin in NYDB:
In aseptic 50mL centrifuge tube, add NYDB and clavacin solution, then add Rhodosporidium paludigenum Fell & Tallman bacteria suspension; Make the ultimate density of thalline reach 1 * 10
5cFU/mL, 8 layers of gauze parcel mouth of pipe with sterilizing, are positioned in 25 ℃ of shaking tables and cultivate, and rotating speed is 180rpm.
Specific as follows:
The clavacin mark product of 5mg are dissolved in 20mL ethyl acetate, draw 5mL and dry up in Nitrogen evaporator, be dissolved in 2.5mLNYDB, make clavacin solution.To adding 100 μ L clavacin solution (i.e. 50 μ g clavacins, its ultimate density is 10ppm) and 100 μ L concentration in the NYDB of 4.8mL, be 5 * 10
6the Rhodosporidium paludigenum Fell & Tallman bacteria suspension of CFU/mL, mixes, thereby makes the ultimate density of thalline reach 1 * 10
5cFU/mL.Not add the blank that is treated to of Rhodosporidium paludigenum Fell & Tallman bacteria suspension.Each processing and blank respectively establish three parallel.The time point detecting is respectively 1 day, 2 days, 3 days, 4 days.
The detection method of clavacin is as follows:
1) sample treatment: after drawing 5mL and cultivating, gains enter in 50mL centrifuge tube, add 5mL ethyl acetate in every pipe, cover the pipe lid of coupling is vibrated 10 minutes in the little shaking table of 250rpm, then in the centrifuge of 3000rpm centrifugal 3 minutes.Draw 3mL supernatant, nitrogen dries up, and is dissolved in (with second acid for adjusting pH value to 4.0) in 1mL deionized water, after the membrane filtration with 2.5 μ m, places, and carries out HPLC detection.
2) HPLC detects: detection method is as follows,
Mobile phase is 10% acetonitrile+90% water (for volume %), and flow velocity is 0.75mL/min, and detection wavelength is 276nm, 35 ℃ of column temperatures, sample size 50 μ L.Selected pillar is 250mm * 4.6mm(internal diameter), the C18 post of particle diameter 5 μ m.In experiment, the appearance time of clavacin is 11min.
More than experiment repeats 3 times.
Experimental result is as Fig. 1.Through the degradation experiment of 4 days, in processed group, the content of clavacin was along with the time constantly declines.Through the cultivation of 1 day, there is 29.79% clavacin be degraded (that is, clavacin content is only 35.1 μ g after testing); Arrived second day, clavacin be almost degraded completely (degradation rate reaches 98.55%, that is, clavacin content is only 0.725 μ g after testing); Within the 4th day, can't detect the existence of clavacin.Meanwhile, the content of clavacin in blank keeps relative stability.
By above-mentioned experiment, we can obtain drawing a conclusion: the clavacin that Rhodosporidium paludigenum Fell & Tallman bacteria suspension of the present invention can effectively be degraded in NYDB.
Clavacin in embodiment 2, Rhodosporidium paludigenum Fell & Tallman bacteria suspension degraded cider (pH is 2.9～3.3), carries out following steps successively:
1), the preparation of bacteria suspension:
With embodiment 1.
2), the degraded of patulin in apple juice:
In aseptic 50mL centrifuge tube, add commercial cider (pH is 2.9～3.3) 4.9ml, record in advance the clavacin that this cider contains 120 μ g/L; Add again Rhodosporidium paludigenum Fell & Tallman bacteria suspension 0.1ml; Make the ultimate density of thalline reach 1 * 10
5cFU/mL, 8 layers of gauze parcel mouth of pipe with sterilizing, are positioned in 25 ℃ of shaking tables and cultivate, and rotating speed is 180rpm.
All the other contents are equal to the step 2 of embodiment 1).
Experimental result as shown in Figure 2.Through the degraded of three days, the content of patulin in apple juice was along with the time constantly declines.Through the cultivation of a day, there is 31.08% clavacin to be degraded; Arrived second day, clavacin over half be degraded (degradation rate reaches 65.49%); Within the 3rd day, can't detect the existence of clavacin.Meanwhile, the content of clavacin in blank keeps relative stability.
By above-mentioned experiment, we can obtain drawing a conclusion: the clavacin that Rhodosporidium paludigenum Fell & Tallman bacteria suspension of the present invention can effectively be degraded in cider.
Illustrate: in the cider that Rhodosporidium paludigenum Fell & Tallman bacteria suspension of the present invention can effectively be applied to be polluted by clavacin, thereby can carry out actual being applicable.
Degradation temperature in comparative example 1, adjustment embodiment 1, degradation temperature drops to 15 ℃ by 25 ℃, and detection time, point was 24h, and all the other are equal to embodiment 1.
Degradation temperature in comparative example 2, adjustment embodiment 1, degradation temperature rises to 35 ℃ by 25 ℃, and detection time, point was 24h, and all the other are equal to embodiment 1.
Comparative example 3, only by embodiment 1 step 2) in the pH value of NYDB culture medium used by 6.0, rise to 8.0, detection time, point was 24h, all the other are equal to embodiment 1.
Remarks explanation: the NYDB culture medium in step 1) is not changed, and its pH value is still 6.0.
Clavacin initial concentration in comparative example 4, adjustment embodiment 1, rises to 30ppm by 10ppm, and detection time, point was 24h, and all the other are equal to embodiment 1.
Clavacin initial concentration in comparative example 5, adjustment embodiment 1, drops to 1ppm by 10ppm, and detection time, point was 24h, and all the other are equal to embodiment 1.
Comparative example 6, by Rhodosporidium paludigenum Fell & Tallman, make the thalline for degrading in embodiment 1 into rhodotorula that background technology is informed, the ultimate density of thalline is constant, is still 1 * 10
5cFU/mL; All the other are equal to embodiment 1.
Comparative example 7, by Rhodosporidium paludigenum Fell & Tallman, make the thalline for degrading in embodiment 1 into Pichia pastoris that background technology is informed, the ultimate density of thalline is constant, is still 1 * 10
5cFU/mL; All the other are equal to embodiment 1.
Comparative example 8,
Only make NYDB used in the preparation process of the step 1) bacteria suspension of embodiment 1 into conventional NYDB culture medium, that is,
Conventional NYDB:10g glucose+8g beef extract+5g yeast extract powder+1L distilled water, then carries out conventional autoclaving.
The Rhodosporidium paludigenum Fell & Tallman bacteria suspension of gained is called bacteria suspension I, in this bacteria suspension I of every ml, contains 5 * 10
6the Rhodosporidium paludigenum Fell & Tallman thalline of CFU.
With this bacteria suspension I, carry out step 2) degraded of clavacin in described NYDB, make equally the ultimate density of thalline reach 1 * 10
5cFU/mL.Remarks explanation: step 2), NYDB used is with the step 2 of embodiment 1).
All the other contents are with embodiment 1.
Above-mentioned all comparative examples are carried out to the detection test of clavacin degradation rate according to the method described in embodiment 1, acquired results is as shown in table 1 below.
The clavacin degradation rate of table 1, different disposal
| ? | Embodiment 1 | Comparative example 1 | Comparative example 2 | Comparative example 3 | Comparative example 4 |
| Clavacin degradation rate | 29.79% | 10.18% | 7.08% | 9.55% | 29.52% |
| ? | Comparative example 5 | Comparative example 6 | Comparative example 7 | Comparative example 8 | ? |
| Clavacin degradation rate | 28.41% | 20.3% | 18.55 | 21.2% | ? |
By above-mentioned comparative example, can be found out, regardless of the initial concentration of clavacin in contaminated thing, the red winter spore yeast of identical final concentration there is no significant difference to the efficiency of its degraded.
Safety experiment:
For detecting the security of the final catabolite of embodiment 1 gained, carried out following experiment:
1), clavacin catabolite obtains
A certain amount of clavacin is added in the 50mL centrifuge tube that 6mL NYDB is housed, makes the ultimate density of clavacin reach respectively 200ppm, 100ppm, 20ppm, 2ppm, 1ppm, 0ppm, each processing have two parallel.Add Rhodosporidium paludigenum Fell & Tallman, make its ultimate density reach 10
5cFU/mL cultivates 3 days in the shaking table of 25 ℃ of 180rpm.By medium centrifugal, results supernatant, mixes the parallel sample of same processed group, obtains the product (primary quantity of its clavacin is respectively 200ppm, 100ppm, 20ppm, 2ppm, 1ppm, 0) by red winter spore yeast degradation clavacin.From different disposal group, draw respectively 400 μ L supernatants, with HPLC, detect, guarantee that clavacin is degradable.
Meanwhile, preparation clavacin solution is dissolved in NYDB, makes its concentration reach respectively 200ppm, 100ppm, 20ppm, 2ppm, 1ppm, 0ppm.
2), the toxicity of clavacin catabolite detects
The Escherichia coli (E.coli) that activated for two generations are centrifugal, obtain thalline and be scattered in fresh LB culture medium and (regulate its concentration, with after one times of NYDB dilution, to make it at OD it
600the light absorption value at place is 0.3 left and right), get the supernatant of yeast degradation clavacin and the E.coli bacteria suspension of 120 μ L (in fact the content of clavacin is 100ppm, 50ppm, 10ppm, 1ppm, 0.5ppm, 0) in 96 aseptic orifice plates of 120 μ L, be put in the constant incubator of 37 ℃.Each processing arranges 6 parallel (i.e. 6 holes), and the E.coli bacteria suspension that the NYDB culture medium of take adds 120 μ L is contrast.Meanwhile, get the clavacin solution of 120 μ L variable concentrations and the E.coli bacteria suspension of 120 μ L, test bar aspergillin itself is to colibacillary toxicity.With ELIASA, detect it in the absorbing wavelength at 600nm place again.Detection time point is 0,1h, 2h, 4h, 6h, 8h, 10h.
Experimental result is as described in Fig. 3 and Fig. 4.From experimental result:
Clavacin has suppressed colibacillary growth, and clavacin concentration is higher, and it is stronger to colibacillary inhibitory action; And the catabolite of clavacin is to colibacillary growth unrestraint effect.By figure, learnt, catabolite can be used as nutrient utilization by Escherichia coli, so Escherichia coli, under the catabolite of high concentration, grow better on the contrary.
Finally, it is also to be noted that, what more than enumerate is only a specific embodiment of the present invention.Obviously, the invention is not restricted to above embodiment, can also have many distortion.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention, all should think protection scope of the present invention.
Claims (4)
1. utilize the method for red winter spore yeast bio degraded clavacin, it is characterized in that: the bacteria suspension of Rhodosporidium paludigenum Fell & Tallman is put into the liquid object of being polluted by clavacin, until the final concentration of Rhodosporidium paludigenum Fell & Tallman is 0.9～1.1 * 10
5cFU/mL, in the shaking table of 150～250rpm in 24～26 ℃ of degradation treatment 2～4 days; Thereby realize the degraded of clavacin.
2. the method for utilizing red winter spore yeast bio degraded clavacin according to claim 1, is characterized in that:
The pH < 7 of the described liquid object of being polluted by clavacin.
3. the method for utilizing red winter spore yeast bio degraded clavacin according to claim 2, is characterized in that:
The preparation method of the bacteria suspension of described Rhodosporidium paludigenum Fell & Tallman is:
From NYDA medium slant, the colony inoculation of picking Rhodosporidium paludigenum Fell & Tallman is in NYDB nutrient solution, under 24～26 ℃, the condition of 150～250rpm, activates; Then be adjusted in the Rhodosporidium paludigenum Fell & Tallman bacteria suspension of every ml and contain 4～6 * 10
6the Rhodosporidium paludigenum Fell & Tallman thalline of CFU;
The formula of NYDA culture medium is: 10g glucose+8g beef extract+5g yeast extract powder+20g agar+1L distilled water;
The formula of NYDB nutrient solution is: 10g glucose+8g beef extract+5g yeast extract powder+10g sodium chloride+1L distilled water; Regulate pH value to 6.0.
4. the method for utilizing red winter spore yeast bio degraded clavacin according to claim 3, is characterized in that:
Described activation is included in the first generation in NYDB nutrient solution and cultivates 24h, and the second generation is cultivated 36h.
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