CN102578676B - Application of Candida guilliermondii strain in elimination of patulin - Google Patents
Application of Candida guilliermondii strain in elimination of patulin Download PDFInfo
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- CN102578676B CN102578676B CN201210045426XA CN201210045426A CN102578676B CN 102578676 B CN102578676 B CN 102578676B CN 201210045426X A CN201210045426X A CN 201210045426XA CN 201210045426 A CN201210045426 A CN 201210045426A CN 102578676 B CN102578676 B CN 102578676B
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- claviformin
- guilliermondii
- patulin
- candida guilliermondii
- monilia
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses application of a yeast strain to elimination of patulin. The invention provides application of Candida guilliermondii to elimination of patulin. Experiments prove that Candida guilliermondii can quickly remove high concentration patulin in culture solution; patulin residues in three concentrations of culture solution can not be detected two days after the Candida guilliermondii cells with concentration being 2*105cfu/ml are inoculated into the culture solution containing 50mu g/ml of patulin, the culture solution containing 100mu g/ml of patulin and the culture solution containing 200mu g/ml of patulin respectively to be cultured. Application provided by the invention can provide a new solution to removal of patulin residues in such food as fruit juice.
Description
Technical field
The present invention relates to the application of a kind of yeast strain in removing claviformin, the particularly application of monilia guilliermondii (Candida guilliermondii) in removing claviformin.
Background technology
Claviformin (Patulin) is the secondary metabolite of part Penicillium and aspergillus fungi.It has extensive and strong toxic action to people and animal.Acute toxicity shows as tic, spasm, subcutis oedema, lung enteremia, enteritis, anuria until death; Chronic toxicity can cause stomach ulcer, and has and suppress immunity, teratogenecity and potential carinogenicity etc., also might influence the male sex's Fertility.Claviformin can be water-soluble, ethanol, acetone, ethyl acetate and chloroform, is slightly soluble in ether, benzene, is insoluble to sherwood oil, and chemical property is stable under acidic conditions.
Produce in the fungi of ability having claviformin, the part fungi is plant pathogen, wherein topmost a kind of be Penicilllum expansum germ (Penicillium expansum).This pathogenic bacteria is to cause one of most important pathogenic bacteria of rotting after fruit is adopted in the world wide, and common host comprises apple, pears, grape, cherry, peach, strawberry etc.It can be to a large amount of claviformin of secretion in the fruit tissue when causing fruit rot.In the fruit juice secondary industry, if the fruit that rots is sneaked in the starting material, the claviformin that the fruit juice of producing so just has in various degree is residual, and that gives human consumer (particularly children) healthyly brings harm.Based on the potential hazard of claviformin to HUMAN HEALTH, a lot of countries all limit the quantity of to the maximum of claviformin in the fruit juice product and stipulate in the world, to limit the quantity of be 50 μ g/kg to the maximum of claviformin in EU, the USFDA regulation fruit juice product, and the intake of WHO suggestion people claviformin every day is no more than 0.4 μ g/kg body weight.
Because it is acid that fruit juice is generally, and claviformin stable in properties under acidic conditions, in a single day so be difficult to natural decomposition after having polluted claviformin in the fruit juice.At present, mainly remove by physical means such as absorption, microwave treatment, but effect not very good, need the novel claviformin of research and development badly and remove means.
Summary of the invention
The new purposes that the purpose of this invention is to provide a kind of monilia guilliermondii (Candida guilliermondii).
New purposes provided by the present invention is specially the application of monilia guilliermondii (Candida guilliermondii) in removing claviformin.
In one embodiment of the invention, described monilia guilliermondii (Candida guilliermondii) is specially in Chinese common micro-organisms culture presevation administrative center and is numbered 2.63 monilia guilliermondii (Candida guilliermondii).
In described application, when described monilia guilliermondii (Candida guilliermondii) CGMCC No.2.63 is initial with the example reaction that contains claviformin, described monilia guilliermondii (Candida guilliermondii) CGMCC No.2.63 is 1000cfu with the ratio range of waiting to remove claviformin: 1 μ g to 4000cfu: 1 μ g, and specifically as 1000cfu: 1 μ g, 2000cfu: 1 μ g or 4000cfu: 1 μ g.
When the described sample that contains claviformin was liquid, the concentration of claviformin was 50-200 μ g/mL in the described sample that contains claviformin, as 50 μ g/mL, 100 μ g/mL or 200 μ g/mL.
In described application, described monilia guilliermondii (Candida guilliermondii) CGMCC No.2.63 and contain the sample of claviformin 25-28 ℃ of reaction, as 26 ℃.
In one embodiment of the invention, the described sample that contains claviformin is specially the nutrient solution that contains claviformin, as 2 * NYDB liquid nutrient medium.The pH of described 2 * NYDB liquid nutrient medium is 5.4, solute is glucose, soy peptone, extractum carnis and yeast extract, solvent is water, the final concentration of described glucose is 20g/L, the final concentration of described soy peptone is 10g/L, the final concentration of described extractum carnis is 2g/L, and the final concentration of described yeast extract is 15g/L.
Described monilia guilliermondii (Candida guilliermondii) CGMCC No.2.63 and the described time of containing the example reaction of claviformin were at least 2 days.In one embodiment of the invention, the described time was specially 2 days, 4 days or 6 days.
In using the process that monilia guilliermondii (Candida guilliermondii) CGMCC No.2.63 removes claviformin, relate in the reaction solution of detection after with described monilia guilliermondii (Candida guilliermondii) CGMCC No.2.63 and the described example reaction that contains claviformin whether the residual step of claviformin is arranged.
The residual concrete grammar of above-mentioned test bar aspergillin can be thin layer chromatography.
In one embodiment of the invention, the used chromatoplate of described thin layer chromatography is specially precoated plate; Used developping agent is specially the mixed solution of toluene, ethyl acetate and 90% aqueous formic acid (volume ratio is 5: 4: 1); Used developer is specially the MBTHHClH of 0.5% (0.5g/100ml)
2O (3-methyl-2 benzothiazolone hydrazone hydrochloride hydrate).Whether in described nutrient solution have claviformin residual concrete grammar be: residual as if orange-yellow reflecting point occurring at described chromatoplate, then illustrate that described claviformin is arranged in the described nutrient solution if judging; Otherwise, illustrate that then not have described claviformin in the described nutrient solution residual.
Experiment showed, monilia guilliermondii (Candida guilliermondii) CGMCC No.2.63 can be in containing the nutrient solution of claviformin fast breeding, and can remove the claviformin of nutrient solution middle and high concentration fast, be 2 * 10 with concentration
5The monilia guilliermondii of cfu/ml (Candida guilliermondii) CGMCC No.2.63 cell is inoculated into respectively in the nutrient solution that contains 50 μ g/ml, 100 μ g/ml and 200 μ g/ml claviformins to be cultivated, and the nutrient solution of three concentration all detects less than claviformin residual after 2 days.Therefore, application provided by the present invention will provide new solution for residual the removing of claviformin in the food such as fruit juice.
Description of drawings
Fig. 1 is that claviformin is to the analytical results of monilia guilliermondii (Candida guilliermondii) CGMCC No.2.63 growth effect.Wherein, ordinate zou is yeast strain concentration.
Fig. 2 is the analytical results that claviformin is removed in the nutrient solution of monilia guilliermondii (Candida guilliermondii) CGMCC No.2.63.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
Monilia guilliermondii (Candida guilliermondii) is to obtain strain number: 2.63 from Institute of Microorganism, Academia Sinica.Yeast (Candida guilliermondii) CGMCC No.2.63 bacterium colony is oyster white, smooth surface, and there is fold at the edge, well-grown on the NYDA substratum.
NYDA substratum (flat board): glucose 10, soy peptone 5, extractum carnis 1, yeast extract 7.5, agar 18, unit: g/L; PH5.5.
NYDB liquid nutrient medium: glucose 10, soy peptone 5, extractum carnis 1, yeast extract 7.5, unit: g/L; PH5.5.
2 times of concentration NYDB liquid nutrient mediums: glucose 20, soy peptone 10, extractum carnis 2, yeast extract 15, unit: g/L; PH5.4.
The influence that embodiment 1, claviformin are grown to monilia guilliermondii (Candida guilliermondii) CGMCC No.2.63
1, the activation of monilia guilliermondii (Candidaguilliermondii) CGMCC No.2.63 bacterial classification
Get monilia guilliermondii (Candida guilliermondii) the CGMCC No.2.63 of-20 ℃ of preservations, it is streak culture on the NYDA flat board.26 ℃ cultivate 72 hours after, the picking yeast cell was inoculated into the NYDB liquid nutrient medium shaking culture 24 hours from the single bacterium colony of well-grown monilia guilliermondii (Candida guilliermondii), 26 ℃ of culture temperature, 200 rev/mins of shaking speed.
2, the preparation of claviformin standard substance mother liquor
Described claviformin is the claviformin standard substance, purchases in Qingdao and gives birth to worker company, is mixed with concentration when using and is the mother liquor of 1mg/ml.Compound method is: accurately takes by weighing 10mg claviformin standard substance, is dissolved in the 10ml distilled water, and abundant stirring and dissolving, and use 0.22 μ m millipore filtration to carry out filtration sterilization.
3, contain the preparation of the nutrient solution of different concns claviformin
Use NYDB nutrient solution, claviformin standard substance mother liquor and the sterilized water of 2 times of concentration, make according to the proportioning in the table 1 and contain 0 μ g/ml respectively, 50 μ g/ml, the nutrient solution of the claviformin of 100 μ g/ml and 200 μ g/ml.
Table 1 contains the prescription of the nutrient solution of different concns claviformin
| Claviformin concentration (μ g/ml) | 0 | 50 | 100 | 200 |
| Claviformin standard substance mother liquor (ml) | 0 | 0.1 | 0.2 | 0.4 |
| 2 times of concentration NYDB nutrient solutions (ml) | 1 | 1 | 1 | 1 |
| Sterilized water (ml) | 1 | 0.9 | 0.8 | 0.6 |
4, claviformin is to the analysis of monilia guilliermondii (Candida guilliermondii) CGMCC No.2.63 growth effect
What will be inoculated into step 3 preparation through monilia guilliermondii (Candida guilliermondii) the CGMCC No.2.63 of overactivation contains 0 μ g/ml respectively, 50 μ g/ml, in the nutrient solution of 100 μ g/ml and 200 μ g/ml claviformins, making the cell concn of inoculation back monilia guilliermondii (Candida guilliermondii) CGMCC No.2.63 is 2 * 10
5Cfu/ml, 26 ℃ of culture temperature, 200 rev/mins of shaking speed, each is handled 3 times and repeats.After the inoculation, be cultured to the 1st, 2,3,4 respectively, from each is handled, taking out 20 μ l cell culture fluids in the time of 5,6 days, calculating cell number with after 10 times of the sterilized water dilutions with blood counting chamber, and obtain cell concn that each is handled through converting.
The result as shown in Figure 1, claviformin in the nutrient solution has certain influence to the propagation of monilia guilliermondii (Candida guilliermondii) CGMCC No.2.63, yet does not change the trend of monilia guilliermondii (Candida guilliermondii) CGMCC No.2.63 fast breeding.Cultivate after 1 day, contain each monilia guilliermondii (Candida guilliermondii) CGMCC No.2.63 cell concn of handling of claviformin and reach about 4 * 10
8Cfu/ml; Cultivate after 6 days, each cell concn of handling is all above 1 * 10
9Cfu/ml.
The detection of the removing ability of embodiment 2, the claviformin of monilia guilliermondii (Candida guilliermondii) CGMCC No.2.63
1, the activation of bacterial classification
With embodiment 1 step 1.
2, the preparation of claviformin standard substance mother liquor
With embodiment 1 step 2.
3, contain the preparation of the nutrient solution of different concns claviformin
With embodiment 1 step 3.
4, the analysis of the removing ability of the claviformin of monilia guilliermondii (Candida guilliermondii) CGMCC No.2.63
What will be inoculated into step 3 preparation through yeast (Candida guilliermondii) the CGMCC No.2.63 of overactivation contains 50 μ g/ml respectively, in the nutrient solution of 100 μ g/ml and 200 μ g/ml claviformins, making the cell concn of inoculation back monilia guilliermondii (Candida guilliermondii) is 2 * 10
5Cfu/ml, 26 ℃ of culture temperature, 200 rev/mins of shaking speed, each is handled 3 times and repeats.Simultaneously, the claviformin treatment group of each different concns all arranges a contrast that does not add monilia guilliermondii (Candida guilliermondii) CGMCC No.2.63.After the inoculation, be cultured to the 2nd day respectively, from handling, each takes out 50 μ l cell culture fluids when the 4th day and the 6th day, centrifugal 5 minutes of 10000g, get supernatant and detect residual claviformin in the nutrient solution with thin layer chromatography (TLC), simultaneously working concentration is respectively 50 μ g/ml, the claviformin standard substance aqueous solution of 100 μ g/ml and 200 μ g/ml in contrast, concrete detection method is as follows:
The activation of chromatoplate: the chromatoplate that uses is precoated plate, and specification is 10cm * 10cm.Carry out no sample with developping agent earlier before using and launch, then 120 ℃ of bakings 1 hour.Cooling back is with the pencil line that scores marks at each 1cm place of distance up-and-down boundary.
Point sample: on chromatoplate, the point sample baseline is apart from base 1.0cm with the glass capillary point sample.
Launch: the developping agent that uses is 5: 4: 1 as the mixed solution of toluene, ethyl acetate and 90% aqueous formic acid, three's volume ratio.The chromatoplate of having put sample put into the chromatography cylinder that adds developping agent in, the degree of depth that immerses developping agent be apart from about the 0.5cm of chromatoplate base, builds sheet glass, when waiting to be expanded to the scribing position place of institute's mark, the taking-up chromatoplate dries.
Colour developing: the developer of use is the MBTHHClH of 0.5% (0.5g/100ml)
2O (3-methyl-2 benzothiazolone hydrazone hydrochloride hydrate).On chromatoplate, evenly spray earlier developer, then 120 ℃ of bakings 15 minutes, be chilled to room temperature after, the position of claviformin on chromatoplate should be orange color dot.
Observations: reference standards, according to the removing ability of assessing the claviformin of monilia guilliermondii (Candida guilliermondii) CGMCC No.2.63 with size that has or not of colour developing point.If orange-yellow reflecting point occurs, then illustrate and contain claviformin in the counter sample, and orange-yellow reflecting point is more big shows that the content of claviformin in the counter sample is more high.For the claviformin nutrient solution of having inoculated yeast strain, orange-yellow reflecting point size illustrates then that more near the control group of corresponding not inoculation yeast bacterial strain the removing ability of the claviformin of (Candida guilliermondii) the CGMCC No.2.63 of monilia guilliermondii in this group is more poor.
The result as shown in Figure 2, inoculating monilia guilliermondii (Candida guilliermondii) after CGMCC No.2.632 days, each is handled and all detects residual less than claviformin in the nutrient solution, and does not inoculate residual (the corresponding standard control group of each treatment group also can detect the residual of claviformin significantly) that can detect claviformin in the control treatment of monilia guilliermondii (Candida guilliermondii) CGMCC No.2.63 significantly.
Claims (1)
- Monilia guilliermondii ( Candida guilliermondii) application in removing claviformin;When described monilia guilliermondii and the nutrient solution that contains claviformin reacted initial, described monilia guilliermondii was 1000cfu:1 μ g to 4000cfu:1 μ g with the proportioning of waiting to remove claviformin;Described monilia guilliermondii and contain the nutrient solution of claviformin 25-28 ℃ of reaction;The concentration of claviformin is 50-200 μ g/mL in the described nutrient solution that contains claviformin.
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| CN104312924A (en) * | 2014-06-30 | 2015-01-28 | 陕西师范大学 | Byssochlamys nivea FF1-2, and screening method and application thereof |
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| CN104403973B (en) * | 2014-12-05 | 2018-02-23 | 江南大学 | It is a kind of that there is the digestion lactobacillus for removing patulin effect and its application |
| CN106243205B (en) * | 2016-08-17 | 2019-08-02 | 中国科学院植物研究所 | A kind of albumen of clavacin of degrading and its encoding gene and application |
| CN109499039B (en) * | 2018-11-21 | 2020-07-14 | 中国科学院植物研究所 | L application of uria-Bertani culture medium in degradation of patulin |
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Non-Patent Citations (7)
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| CASTORIA R.等.Effect of the biocontrol yeast Rhodotorula glutinis Strain LS11 on patulin accumulation in stored apples.《Phytopathology》.2005,第95卷(第11期),1271-1278. |
| COELHO A R.等.Penicillium expansum versus antagonist yeasts and patulin degradation in vitro.《Brazilian Archives of Biology and Technology》.2007,第50卷(第7期),725-733. |
| Effect of biocontrol agents Candida sake and Pantoea agglomerans on Penicillium expansum growth and patulin accumulation in apples;MORALES H.等;《International Journal of Food Microbiology》;20081231;第122卷(第1-2期);摘要、第3.2节 * |
| Effect of the biocontrol yeast Rhodotorula glutinis Strain LS11 on patulin accumulation in stored apples;CASTORIA R.等;《Phytopathology》;20051231;第95卷(第11期);1271-1278 * |
| JP特开平4-360669A 1992.12.14 |
| MORALES H.等.Effect of biocontrol agents Candida sake and Pantoea agglomerans on Penicillium expansum growth and patulin accumulation in apples.《International Journal of Food Microbiology》.2008,第122卷(第1-2期),摘要、第3.2节. |
| Penicillium expansum versus antagonist yeasts and patulin degradation in vitro;COELHO A R.等;《Brazilian Archives of Biology and Technology》;20071231;第50卷(第7期);725-733 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104312924A (en) * | 2014-06-30 | 2015-01-28 | 陕西师范大学 | Byssochlamys nivea FF1-2, and screening method and application thereof |
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