CN112244202A - Application of cryptococcus for controlling degradation of ochratoxin A in grape juice - Google Patents
Application of cryptococcus for controlling degradation of ochratoxin A in grape juice Download PDFInfo
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- CN112244202A CN112244202A CN202010907991.7A CN202010907991A CN112244202A CN 112244202 A CN112244202 A CN 112244202A CN 202010907991 A CN202010907991 A CN 202010907991A CN 112244202 A CN112244202 A CN 112244202A
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- A—HUMAN NECESSITIES
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Abstract
The invention belongs to the technical field of biological control, and relates to application of cryptococcus rhodochrous in degradation control of ochratoxin A in grape juice. The method comprises the following steps: firstly, preparing a bacterial suspension; inoculating cryptococcus Y3 into an NYDB culture medium, and culturing and activating under the shaking conditions of 28-30 ℃ and 180 rpm; then coating the activated bacterial liquid on an NYDA culture medium, and culturing at 28-30 ℃ for 48-60 h; then, single colony was selected and inoculated into PM medium for culturing for 48 hours, after culturing, the bacterial cell was obtained by centrifugation, washed with sterile distilled water, and diluted to 1X 108cell/mL of bacterial suspension; the ochratoxin A is inoculated into the grape juice according to the volume ratio of 1-2 percent, so that the purpose of controlling and degrading the ochratoxin A in the grape juice can be realized; the method is safe and environment-friendly, can efficiently control ochratoxin A in grape juice, and can be used for 3 daysThe ochratoxin A in the grape juice is completely degraded.
Description
Technical Field
The invention belongs to the technical field of biological control, and particularly relates to application of cryptococcus rhodochrous in degradation control of ochratoxin A in grape juice.
Background
Ochratoxin a (OTA) is a fungal secondary metabolite that is second only toxic to aflatoxins. The toxin has a molecular formula of C20H18O6CIN with molecular weight of 404 g/mol. Ochratoxin A is isocoumarin, which is prepared from 7-carboxy-5-chloro-8-hydroxy-3, 4-dihydro-3-R-methylisocoumarin (ochratoxin alpha) and L-beta-phenylalanine through amide bond. Ochratoxin A is not readily soluble in water, is very soluble in organic solvents and NaHCO3The solution is resistant to acid environment and is not easy to degrade under high temperature condition, and the baking environment can degrade partial toxin.
International cancer research institute and world health organization generalize ochratoxin a, a human carcinogen of class 2B, which has nephrotoxicity, hepatotoxicity, carcinogenic teratogenic mutation, immunotoxicity, genotoxicity, etc., especially in association with kidney disease in the bardry and crohn's regions, and is a serious threat to human and animal health. In addition, according to various reports, the toxin pollution range is wide, and the toxin can be detected in various foods and feeds. The pathogenic bacteria that produce ochratoxin a primarily are the Penicillium (Penicillium) and Aspergillus (Aspergillus) fungi. Ochratoxin a is first found in corn and then detected in various grains (wheat, rice, etc.), grapes and their products (wine, grape juice), soy sauce, nuts, meat products, coffee, cheese, etc. Wherein, the grapes and the products thereof are the main pollution objects of OTA. Because of the wide pollution range and high toxicity of the toxin, countries all over the world make strict regulations on the residual quantity of the toxin in food: the world health organization recommends a daily tolerated intake of 5ng OTA/kg BW/day; the European Union stipulates that the maximum residual quantity of ochratoxin A in grains, wine and raisins respectively cannot exceed 5 mu g/kg, 2 mu g/kg and 10 mu g/kg; the content of ochratoxin A in the wine is not more than 2.0 mu g/kg according to the international grape and wine organization rules; GB2761-2017 and GB2762-2017 specify that the limit standard of ochratoxin A in wine is 2.0 mu g/kg, the limit standard of ochratoxin A in coffee beans and coffee powder is 5.0 mu g/kg, and the limit standard of ochratoxin A in instant coffee is 10.0 mu g/kg.
The existing method for controlling and degrading ochratoxin A comprises a physical mode, a chemical mode and a biological mode. Physical degradation of ochratoxin a involves adsorption and degradation. Adsorption is a common method for removing toxins, common adsorbents comprise bentonite, pumice, sepiolite, montmorillonite, activated carbon, yeast cell walls and nano materials, and although adsorption can remove toxins efficiently, adsorption cannot completely degrade toxins, so that the toxins are left in the environment to cause secondary pollution; heating is also an effective way to remove ochratoxin A, but is easy to cause the problem of product quality reduction; in addition, ochratoxin A can be degraded by means of alkaline environment, ozone, extrusion processing and the like. Chemical degradation of ochratoxin A is also a common detoxification mode, and the chemical degradation of ochratoxin A has been researched to be diversified, and catalase (POD) is a general substance for degrading aflatoxin, vomitoxin and ochratoxin A. POD extracted from rice bran, rat liver microsomes, and commercial enzyme POD were both effective in degrading ochratoxin A. Protein and sugar are another effective substance for degrading ochratoxin A, and researches show that the addition of reducing sugar during baking is helpful for degrading ochratoxin A in oat, but the method is complex, and the influence on food is determined, such as whether the addition amount and type of the reducing sugar in other food affect the flavor, appearance and the like of the food, increase the possibility of food spoilage and the like.
The biological detoxification technology is to degrade toxins in feed and food through microorganisms such as bacteria or yeasts and enzyme substances secreted by the microorganisms, and the method controls the toxins to be safe and pollution-free, thereby being a promising direction.
Disclosure of Invention
Aiming at the defects and shortcomings of the prior art, the invention provides Cryptococcus podzuvine Y3 which is selected from soil and used as a separating sieve, has obvious degradation control effect on ochratoxin A in grape juice and has potential application value; the cryptococcus rhodochrous Y3 used in the invention is determined to have high safety and no harm to human body by mouse acute toxicity test.
The cryptococcus yeast system for controlling and degrading ochratoxin A provided by the invention is screened and separated from soil collected from orchards in the sentence-volume city of Zhenjiang, and is cultured on an NYDA solid medium plate at 28 ℃ for 3d for morphological observation; the 5.8S rDNA-ITS region sequence of this strain has been analyzed and identified molecularly.
The yeast strain for controlling and degrading ochratoxin A in grape juice provided by the invention is currently preserved in China Center for Type Culture Collection (CCTCC) of Wuhan university in Wuhan, China, and the preservation number is as follows: CCTCC NO: m2017505, the proposed classification was named Cryptococcus (Cryptococcus podzolicus) Y3. In addition, the applicant of the strain utilized in the present invention has applied for chinese patent invention, CN201711183834.0, with the name: a yeast for degrading citrinin and application thereof.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
(1) preparing a bacterial suspension: inoculating cryptococcus rhodochrous Y3 into an NYDB culture medium, culturing for 20-24 h under the shaking conditions of 28-30 ℃ and 180rpm, and activating to obtain activated bacterial liquid; then coating the activated bacterial liquid on an NYDA solid culture medium, and culturing at 28-30 ℃ for 48-60 h; then selecting single colony of cryptococcus rhodochrous Y3 growing in NYDA solid culture medium, inoculating into PM culture medium, culturing, centrifuging to obtain thallus, washing with sterile distilled water for several times, and diluting with sterile water to 1 × 108cell/mL of bacterial suspension;
(2) and (2) inoculating the bacterial suspension prepared in the step (1) into grape juice to realize the purpose of controlling and degrading ochratoxin A in the grape juice.
Preferably, the rotation speed of the centrifugation in the step (1) is 7000r/min, and the time is 10 min.
Preferably, the composition of the NYDA culture medium in the step (1) is as follows: calculated by 1000mL, 8g of beef extract, 5g of yeast extract, 10g of glucose, 20g of agar and the balance of distilled water, and carrying out moist heat sterilization at 115 ℃ for 15 min.
Preferably, the NYDB culture medium in the step (1) comprises the following components: 5g of yeast extract, 10g of glucose, 8g of beef extract and the balance of distilled water in 1000mL, wherein the pH is natural, and the sterilization is carried out for 15min at 115 ℃.
Preferably, the components of the PM medium in the step (1) are as follows: 2g of maltose, 10g of cane sugar, 5g of yeast extract, 5g of peptone and 1000mL of primary distilled water, and sterilizing at 115 ℃ for 15 min.
Preferably, the single colony in the step (1) is inoculated into a PM culture medium for culturing at the temperature of 28-30 ℃ for 48 hours.
Preferably, in the step (1), the washing with sterile distilled water is carried out for 2 to 3 times.
Preferably, the inoculation amount of the bacterial suspension inoculated into the grape juice in the step (2) is 1-2%; namely the bacterial suspension is 1 to 2 percent of the volume of the grape juice.
The control effect is as follows:
will be 1 × 108Inoculating cell/mL bacterial suspension into a PM culture medium or sterile natural grape juice according to the proportion of 2%, and then adding an ochratoxin A standard substance to enable the final concentration to be 1 mu g/mL; performing shake culture at 28 deg.C and 180rpm in a dark environment, and sampling for 5 days at regular time each day; the control group was prepared by replacing the bacterial suspension with an equal amount of sterile water.
The results show that: the yeast can completely degrade ochratoxin A in PM culture medium within 5 days, and can completely degrade ochratoxin A in grape juice within 3 days; the result shows that cryptococcus rhodochrous Y3 has good control and degradation effects on ochratoxin A in a PM culture medium, and has better control effects on ochratoxin A in natural grape juice.
The invention has the advantages that:
(1) the invention provides a yeast strain which is separated from soil and can efficiently degrade ochratoxin A, cryptococcus rhodochrous Y3 can efficiently control and degrade ochratoxin A in grape juice, and ochratoxin A in grape juice can be completely degraded within 3 days.
(2) The cryptococcus rhodochrous Y3 used in the invention is determined to have high safety and no harm to human body through an acute toxicity test of mice, so that the cryptococcus rhodochrous Y3 can be applied to controlling ochratoxin A pollution in food, feed and products thereof and the like, and the edible safety of the food and the products thereof is guaranteed.
Drawings
FIG. 1 is a graph showing the controlled degradation effect of Cryptococcus rhodochrous Y3 on ochratoxin A in PM medium; indicates that there was a significant difference between the treated and control groups at the same measurement time (P < 0.05).
FIG. 2: a controlled degradation effect diagram of cryptococcus rhodochrous Y3 on ochratoxin A in grape juice; indicates that there was a significant difference between the treated and control groups at the same measurement time (P < 0.05).
Note: control is control group; podzolics is cryptococcus Y3 treatment group.
Detailed Description
The invention will be explained in more detail by means of the following examples; the following examples are illustrative only, and the present invention is not limited by these examples.
Example 1:
the controlled degradation effect of cryptococcus rhodochrous Y3 on ochratoxin A in a PM culture medium;
firstly, taking out yeast from minus 80 ℃, activating for 24h through NYDB, sucking 20-30 microliter, coating the yeast on NYDA, culturing for 48-60h, selecting a single colony, inoculating the single colony to a PM (2 g of maltose, 10g of cane sugar, 5g of yeast extract, 5g of peptone and 1000mL of primary distilled water) culture medium, culturing for 48h, centrifuging at 7000rpm for 10min, and cleaning with sterile distilled water to obtain thalli; diluting the thallus with sterile water to 1 × 108cells/mL bacterial suspension is added into PM culture medium according to the addition amount of 2%, and then ochratoxin A standard substance is added in a dark place to enable the final concentration to be 1 mu g/mL. Shaking culture at 28 deg.C under dark condition at 180rpm, and sampling periodically for 5 days. After centrifuging the sample at 10000rpm for 15min, taking out the supernatant, adding equal chromatographic grade methanol, then passing through a 0.22 μm organic pinhole filter head, and measuring by HPLC-FLD.
The liquid phase detection conditions are as follows: agilent1260 is adopted; using a Zorbax SB-C18 column (250 mm. times.4.6 mm, 5 μm); mobile phase: acetonitrile: 1% acetic acid (60:40, v/v); flow rate: 1.0 mL/min; sample introduction amount: 20 μ L, column temperature: 30 ℃; detection wavelength: the excitation wavelength is 333nm, and the emission wavelength is 460 nm; the peak-off time was about 5.4 min.
As can be seen from FIG. 1, the cryptococcus rhodochrous Y3 completely degraded ochratoxin A in the culture medium within 5 days, the degradation effect was relatively slow in the two days before the culture, and the degradation rate was the greatest and then flatter from day 2 to day 3. On day 5 of culture, ochratoxin A was not detected under liquid phase conditions.
Example 2:
the cryptococcus rhodochrous Y3 controls and degrades ochratoxin A in natural grape juice;
the cultivation procedure is as described in example 1, except that 1X 108cells/mL of the bacterial suspension was added to sterile grape juice at 2% addition. Then, ochratoxin A standard substance is added in the dark to enable the final concentration to be 1 mu g/mL. Shaking culture at 28 deg.C under dark condition at 180rpm, and sampling periodically for 5 days. After centrifuging the sample at 10000rpm for 15min, taking out the supernatant, adding equal chromatographic grade methanol, then passing through a 0.22 μm organic pinhole filter head, and measuring by HPLC-FLD.
The liquid phase detection conditions were the same as in example 1;
as can be seen from FIG. 2, Cryptococcus rhodochrous Y3 has a very good controlled degradation effect on ochratoxin A in grape juice and can be completely degraded within 3 days. The yeast has high efficiency of degrading ochratoxin A2 days before culture, the degradation efficiency tends to be stable from 2 days to 3 days, and no ochratoxin A can be detected at 3 days. The juice has a binding effect on the toxins, probably due to conjugation reactions between the toxins and carbohydrates or proteins in the juice. Compared with the culture medium, the cryptococcus rhodochrous Y3 has better degradation effect on ochratoxin A in grape juice, and can realize complete degradation of ochratoxin A in the grape juice.
Description of the drawings: the above embodiments are only used to illustrate the present invention and do not limit the technical solutions described in the present invention; thus, while the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted; all such modifications and variations are intended to be included herein within the scope of this disclosure and the present invention and protected by the following claims.
Claims (8)
1. The application of cryptococcus neoformans for controlling and degrading ochratoxin A in grape juice is characterized by comprising the following specific steps:
(1) preparing a bacterial suspension: inoculating cryptococcus rhodochrous Y3 into an NYDB culture medium, culturing for 20-24 h under the shaking conditions of 28-30 ℃ and 180rpm, and activating to obtain activated bacterial liquid; then coating the activated bacterial liquid on an NYDA solid culture medium, and culturing at 28-30 ℃ for 48-60 h; then selecting single colony of cryptococcus rhodochrous Y3 growing in NYDA solid culture medium, inoculating into PM culture medium, culturing, centrifuging to obtain thallus, washing with sterile distilled water for several times, and diluting with sterile water to 1 × 108cell/mL of bacterial suspension;
(2) and (2) inoculating the bacterial suspension prepared in the step (1) into grape juice to realize the purpose of controlling and degrading ochratoxin A in the grape juice.
2. Use according to claim 1, characterized in that the rotation speed of the centrifugation in step (1) is 7000r/min and the time is 10 min.
3. The use according to claim 1, wherein the NYDA medium in step (1) has the following composition: calculated by 1000mL, 8g of beef extract, 5g of yeast extract, 10g of glucose, 20g of agar and the balance of distilled water, and carrying out moist heat sterilization at 115 ℃ for 15 min.
4. The use according to claim 1, wherein the NYDB medium comprises the following components: 5g of yeast extract, 10g of glucose, 8g of beef extract and the balance of distilled water in 1000mL, wherein the pH is natural, and the sterilization is carried out for 15min at 115 ℃.
5. The use according to claim 1, wherein the PM medium composition in step (1) is as follows: 2g of maltose, 10g of cane sugar, 5g of yeast extract, 5g of peptone and 1000mL of primary distilled water, and sterilizing at 115 ℃ for 15 min.
6. The use according to claim 1, wherein the temperature for inoculating the single colony in the PM medium in the step (1) for culturing is 28-30 ℃ and the time is 48 h.
7. Use according to claim 1, wherein in step (1) the washing with sterile distilled water is carried out several times, in particular 2 to 3 times.
8. Use according to claim 1, characterized in that the bacterial suspension is inoculated in the grape must in step (2) in an amount of between 1% and 2%.
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| CN202010907991.7A CN112244202B (en) | 2020-09-02 | 2020-09-02 | Application of cryptococcus for controlling degradation of ochratoxin A in grape juice |
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| CN202010907991.7A CN112244202B (en) | 2020-09-02 | 2020-09-02 | Application of cryptococcus for controlling degradation of ochratoxin A in grape juice |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040208956A1 (en) * | 2001-12-20 | 2004-10-21 | Gerd Schatzmayr | Microorganism for biological detoxification of mycotoxins, namely ochratoxins and/or zearalenons, as well as method and use thereof |
| CN106472961A (en) * | 2016-10-26 | 2017-03-08 | 北京科润生科技发展有限公司 | The detoxifying agent of ochratoxin and its preparation technology in a kind of biodegradation feedstuff |
| CN107815423A (en) * | 2017-11-23 | 2018-03-20 | 江苏大学 | One plant degraded citrinin saccharomycete and application |
| CN109221895A (en) * | 2018-10-30 | 2019-01-18 | 河南后羿实业集团有限公司 | Feeding biodegradable mycotoxin preparation of one kind and its preparation method and application |
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- 2020-09-02 CN CN202010907991.7A patent/CN112244202B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040208956A1 (en) * | 2001-12-20 | 2004-10-21 | Gerd Schatzmayr | Microorganism for biological detoxification of mycotoxins, namely ochratoxins and/or zearalenons, as well as method and use thereof |
| CN106472961A (en) * | 2016-10-26 | 2017-03-08 | 北京科润生科技发展有限公司 | The detoxifying agent of ochratoxin and its preparation technology in a kind of biodegradation feedstuff |
| CN107815423A (en) * | 2017-11-23 | 2018-03-20 | 江苏大学 | One plant degraded citrinin saccharomycete and application |
| CN109221895A (en) * | 2018-10-30 | 2019-01-18 | 河南后羿实业集团有限公司 | Feeding biodegradable mycotoxin preparation of one kind and its preparation method and application |
Non-Patent Citations (1)
| Title |
|---|
| 王玉萍 等: "微生物对赭曲霉毒素A的生物脱毒机理研究进展", 《农业生物技术学报》 * |
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