CN102578676A - Application of Candida guilliermondii strain to elimination of patulin - Google Patents
Application of Candida guilliermondii strain to elimination of patulin Download PDFInfo
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- CN102578676A CN102578676A CN201210045426XA CN201210045426A CN102578676A CN 102578676 A CN102578676 A CN 102578676A CN 201210045426X A CN201210045426X A CN 201210045426XA CN 201210045426 A CN201210045426 A CN 201210045426A CN 102578676 A CN102578676 A CN 102578676A
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- Prior art keywords
- clavacin
- guilliermondii
- patulin
- candida guilliermondii
- monilia
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Links
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- 230000008030 elimination Effects 0.000 title abstract 3
- 238000003379 elimination reaction Methods 0.000 title abstract 3
- 241001363490 Monilia Species 0.000 claims description 39
- 238000006243 chemical reaction Methods 0.000 claims description 6
- 235000015203 fruit juice Nutrition 0.000 abstract description 8
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- 238000002360 preparation method Methods 0.000 description 6
- 239000002904 solvent Substances 0.000 description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 5
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- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
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- IYXXQOGEFHAQGU-UHFFFAOYSA-N [(3-methyl-1,3-benzothiazol-2-ylidene)amino]azanium;chloride;hydrate Chemical compound O.Cl.C1=CC=C2SC(=NN)N(C)C2=C1 IYXXQOGEFHAQGU-UHFFFAOYSA-N 0.000 description 2
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- FIVPIPIDMRVLAY-UHFFFAOYSA-N aspergillin Natural products C1C2=CC=CC(O)C2N2C1(SS1)C(=O)N(C)C1(CO)C2=O FIVPIPIDMRVLAY-UHFFFAOYSA-N 0.000 description 1
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- FIVPIPIDMRVLAY-RBJBARPLSA-N gliotoxin Chemical compound C1C2=CC=C[C@H](O)[C@H]2N2[C@]1(SS1)C(=O)N(C)[C@@]1(CO)C2=O FIVPIPIDMRVLAY-RBJBARPLSA-N 0.000 description 1
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
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Images
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 clavacin, the particularly application of monilia guilliermondii (Candida guilliermondii) in removing clavacin.
Background technology
Clavacin (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, hypodermis oedema, lung enteremia, enteritis, anuria until death; Chronic toxicity can cause gastric ulcer, and has and suppress immunity, teratogenesis and potential carcinogenic etc., also might influence the male sex's fecundity.Clavacin can be water-soluble, ethanol, acetone, ethyl acetate and chloroform, is slightly soluble in ether, benzene, is insoluble to benzinum, and chemical property is stable under acid condition.
Produce in the fungi of ability having clavacin, the part fungi is a plant pathogen, wherein topmost a kind of be penicillium expansum germ (Penicillium expansum).This pathogen is to cause one of most important pathogen of rotting after fruit is adopted in the world wide, and common host comprises apple, pears, grape, cherry, peach, strawberry or the like.It can secrete a large amount of clavacins in fruit tissue when causing fruit rot.In the fruit juice secondary industry, if the fruit that rots is sneaked in the raw material, the clavacin that the fruit juice of producing so just has in various degree is residual, and that gives consumer (particularly children) healthyly brings harm.Based on the potential hazard of clavacin to health; A lot of in the world countries all limit the quantity of to the maximum of clavacin in the fruit juice product and stipulate; To limit the quantity of be 50 μ g/kg to the maximum of clavacin in EU, the USFDA regulation fruit juice product, and the intake of WHO suggestion people clavacin every day is no more than 0.4 μ g/kg body weight.
Because it is acid that fruit juice is generally, and clavacin stable in properties under acid condition, in a single day so be difficult to natural decomposition after having polluted clavacin in the fruit juice.At present, mainly remove, but effect not very good, need the novel clavacin of research and development badly and remove means through physical means such as absorption, microwave treatment.
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 clavacin.
In one embodiment of the invention, said 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 said application; Said monilia guilliermondii (Candida guilliermondii) CGMCC No.2.63 with contain the example reaction of clavacin when initial; Said monilia guilliermondii (Candida guilliermondii) CGMCC No.2.63 is 1000cfu with the ratio range of waiting to remove clavacin: 1 μ g to 4000cfu: 1 μ g, and specifically like 1000cfu: 1 μ g, 2000cfu: 1 μ g or 4000cfu: 1 μ g.
When the said sample that contains clavacin was liquid, the concentration of clavacin was 50-200 μ g/mL in the said sample that contains clavacin, like 50 μ g/mL, 100 μ g/mL or 200 μ g/mL.
In said application, said monilia guilliermondii (Candida guilliermondii) CGMCC No.2.63 and the sample that contains clavacin are 25-28 ℃ of reaction, as 26 ℃.
In one embodiment of the invention, the said sample that contains clavacin is specially the nutrient solution that contains clavacin, like 2 * NYDB fluid nutrient medium.The pH of said 2 * NYDB fluid nutrient medium is 5.4; Solute is glucose, soy peptone, beef extract and yeast extract; Solvent is a water, and the final concentration of said glucose is 20g/L, and the final concentration of said soy peptone is 10g/L; The final concentration of said beef extract is 2g/L, and the final concentration of said yeast extract is 15g/L.
Said monilia guilliermondii (Candida guilliermondii) CGMCC No.2.63 and the said time of containing the example reaction of clavacin were at least 2 days.In one embodiment of the invention, the said time was specially 2 days, 4 days or 6 days.
In using the process that monilia guilliermondii (Candida guilliermondii) CGMCC No.2.63 removes clavacin, relate in the reactant liquor behind the example reaction that detection contains said monilia guilliermondii (Candida guilliermondii) CGMCC No.2.63 and said clavacin whether the residual step of clavacin is arranged.
The residual concrete grammar of above-mentioned test bar aspergillin can be TLC.
In one embodiment of the invention, the used chromatoplate of said TLC is specially prefabricated board; Used solvent is specially the mixed liquor 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 said nutrient solution have clavacin residual concrete grammar be: residual as if orange-yellow reflecting point on said chromatoplate, occurring, then explain that said clavacin is arranged in the said nutrient solution if judging; Otherwise, explain that then not have said clavacin in the said nutrient solution residual.
Experiment showed, monilia guilliermondii (Candida guilliermondii) CGMCC No.2.63 can be in containing the nutrient solution of clavacin fast breeding, and can remove the clavacin 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 clavacins to be cultivated, and the nutrient solution of three concentration all detects less than clavacin residual after 2 days.Therefore, application provided by the present invention will provide new solution for residual the removing of clavacin in the food such as fruit juice.
Description of drawings
Fig. 1 is the analysis result of clavacin to monilia guilliermondii (Candida guilliermondii) CGMCC No.2.63 growth effect.Wherein, ordinate is a yeast strain concentration.
Fig. 2 is the analysis result that monilia guilliermondii (Candida guilliermondii) CGMCC No.2.63 removes clavacin in the nutrient solution.
The specific embodiment
Employed experimental technique is conventional method like no specified otherwise among the following embodiment.
Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.
Monilia guilliermondii (Candida guilliermondii) is to obtain strain number: 2.63 from Institute of Microorganism, Academia Sinica.Saccharomycete (Candida guilliermondii) CGMCC No.2.63 bacterium colony is a milky, smooth surface, and there is fold at the edge, well-grown on the NYDA culture medium.
NYDA culture medium (flat board): glucose 10, soy peptone 5, beef extract 1, yeast extract 7.5, agar 18, unit: g/L; PH5.5.
NYDB fluid nutrient medium: glucose 10, soy peptone 5, beef extract 1, yeast extract 7.5, unit: g/L; PH5.5.
2 times of concentration NYDB fluid nutrient mediums: glucose 20, soy peptone 10, beef extract 2, yeast extract 15, unit: g/L; PH5.4.
The influence that embodiment 1, clavacin 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, with its cultivation of on the NYDA flat board, ruling.26 ℃ cultivate 72 hours after; The picking yeast cell was inoculated into the NYDB fluid nutrient medium shaken cultivation 24 hours from the single bacterium colony of well-grown monilia guilliermondii (Candida guilliermondii); 26 ℃ of cultivation temperature, 200 rev/mins of shaking speed.
2, the preparation of clavacin standard items mother liquor
Said clavacin is the clavacin standard items, purchases in Qingdao and gives birth to worker company, is mixed with the mother liquor of concentration as 1mg/ml when using.Compound method is: accurately takes by weighing 10mg clavacin standard items, is dissolved in the 10ml distilled water, and abundant stirring and dissolving, and use 0.22 μ m miillpore filter to carry out filtration sterilization.
3, contain the preparation of the nutrient solution of variable concentrations clavacin
Use NYDB nutrient solution, clavacin standard items 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 clavacin of 100 μ g/ml and 200 μ g/ml.
Table 1 contains the prescription of the nutrient solution of variable concentrations clavacin
| Clavacin concentration (μ g/ml) | ?0 | 50 | 100 | 200 |
| Clavacin standard items 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, clavacin 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 clavacins, making the cell concentration of inoculation back monilia guilliermondii (Candida guilliermondii) CGMCC No.2.63 is 2 * 10
5Cfu/ml, 26 ℃ of cultivation 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 concentration that each is handled through converting.
The result is as shown in Figure 1; Clavacin 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 concentration of handling of clavacin and reach about 4 * 10
8Cfu/ml; Cultivate after 6 days, each cell concentration of handling is all above 1 * 10
9Cfu/ml.
1, the activation of bacterial classification
With embodiment 1 step 1.
2, the preparation of clavacin standard items mother liquor
With embodiment 1 step 2.
3, contain the preparation of the nutrient solution of variable concentrations clavacin
With embodiment 1 step 3.
4, monilia guilliermondii (Candida guilliermondii) CGMCC No.2.63 is to the analysis of the removing ability of clavacin
What will be inoculated into step 3 preparation through saccharomycete (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 clavacins, making the cell concentration of inoculation back monilia guilliermondii (Candida guilliermondii) is 2 * 10
5Cfu/ml, 26 ℃ of cultivation temperature, 200 rev/mins of shaking speed, each is handled 3 times and repeats.Simultaneously, the clavacin processed group of each variable concentrations all is provided with 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 each is handled, taking out 50 μ l cell culture fluids, centrifugal 5 minutes of 10000g when the 4th day and the 6th day; Get supernatant and detect residual clavacin in the nutrient solution with TLC (TLC); Working concentration is respectively 50 μ g/ml simultaneously, and the clavacin standard items aqueous solution of 100 μ g/ml and 200 μ g/ml is as contrast, and concrete detection method is following:
The activation of chromatoplate: the chromatoplate that uses is prefabricated board, and specification is 10cm * 10cm.Carry out n.s with solvent earlier before using and launch, then 120 ℃ of bakings 1 hour.Cooling back with pencil at the line that scores marks apart from each 1cm place of up-and-down boundary.
Point sample: on chromatoplate, the point sample baseline is 1.0cm apart from the base with the capillary glass tube point sample.
Launch: the solvent that uses is 5: 4: 1 as the mixed liquor 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 solvent in, the degree of depth that immerses solvent be apart from about the 0.5cm of chromatoplate base, builds glass plate, 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 clavacin on chromatoplate should be orange color dot.
Observed result: reference standards, according to the removing ability to clavacin that has or not of colour developing point with size assessment monilia guilliermondii (Candida guilliermondii) CGMCC No.2.63.If orange-yellow reflecting point occurs, then explain and contain clavacin in the counter sample, and orange-yellow reflecting point shows that more greatly the content of clavacin in the counter sample is high more.For the clavacin nutrient solution of having inoculated yeast strain; The control group of the more approaching corresponding not inoculation yeast bacterial strain of orange-yellow reflecting point size, monilia guilliermondii (Candida guilliermondii) CGMCC No.2.63 was poor more to the removing ability of clavacin during then explanation should be organized.
The result is 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 clavacin in the nutrient solution, and does not inoculate residual (the pairing standard control group of each processed group also can detect the residual of clavacin significantly) that can detect clavacin in the control treatment of monilia guilliermondii (Candida guilliermondii) CGMCC No.2.63 significantly.
Claims (4)
1. the application of monilia guilliermondii (Candida guilliermondii) in removing clavacin.
2. application according to claim 1; It is characterized in that: in the said application; Said monilia guilliermondii (Candida guilliermondii) and contain the example reaction of clavacin when initial, said monilia guilliermondii (Candida guilliermondii) is 1000cfu with the proportioning of waiting to remove clavacin: 1 μ g to 4000cfu: 1 μ g.
3. application according to claim 2 is characterized in that: the concentration of clavacin is 50-200 μ g/mL in the said sample that contains clavacin.
4. according to arbitrary described application among the claim 1-3, it is characterized in that: in the said application, said monilia guilliermondii (Candida guilliermondii) and the sample that contains clavacin are 25-28 ℃ of reaction.
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| CN106243205A (en) * | 2016-08-17 | 2016-12-21 | 中国科学院植物研究所 | The albumen of a kind of clavacin of degrading and encoding gene thereof and application |
| 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 |
| CN109499039A (en) * | 2018-11-21 | 2019-03-22 | 中国科学院植物研究所 | Application of the Luria-Bertani culture medium in degradation clavacin |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104312924B (en) * | 2014-06-30 | 2016-05-04 | 陕西师范大学 | Snow-white silk clothes mould FF1-2 and screening technique and application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH04360669A (en) * | 1991-06-06 | 1992-12-14 | Kanebo Ltd | Preparation of chilled fruit juice drink |
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2012
- 2012-02-24 CN CN201210045426XA patent/CN102578676B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH04360669A (en) * | 1991-06-06 | 1992-12-14 | Kanebo Ltd | Preparation of chilled fruit juice drink |
Non-Patent Citations (3)
| Title |
|---|
| CASTORIA R.等: "Effect of the biocontrol yeast Rhodotorula glutinis Strain LS11 on patulin accumulation in stored apples", 《PHYTOPATHOLOGY》, vol. 95, no. 11, 31 December 2005 (2005-12-31), pages 1271 - 1278 * |
| COELHO A R.等: "Penicillium expansum versus antagonist yeasts and patulin degradation in vitro", 《BRAZILIAN ARCHIVES OF BIOLOGY AND TECHNOLOGY》, vol. 50, no. 7, 31 December 2007 (2007-12-31), pages 725 - 733 * |
| 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》, vol. 122, no. 12, 31 December 2008 (2008-12-31), pages 3 - 2 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN106243205A (en) * | 2016-08-17 | 2016-12-21 | 中国科学院植物研究所 | The albumen of a kind of clavacin of degrading and encoding gene thereof and 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 |
| CN109499039A (en) * | 2018-11-21 | 2019-03-22 | 中国科学院植物研究所 | Application of the Luria-Bertani culture medium in degradation clavacin |
| 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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| CN102578676B (en) | 2013-09-25 |
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