CN103387941A - Identification medium for generating aflatoxin strains and identification method of the same - Google Patents

Identification medium for generating aflatoxin strains and identification method of the same Download PDF

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Publication number
CN103387941A
CN103387941A CN2013102962497A CN201310296249A CN103387941A CN 103387941 A CN103387941 A CN 103387941A CN 2013102962497 A CN2013102962497 A CN 2013102962497A CN 201310296249 A CN201310296249 A CN 201310296249A CN 103387941 A CN103387941 A CN 103387941A
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substratum
medium
aflatoxin
identified
value
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许艳丽
鲍蕾
静平
吴振兴
孙静克
唐静
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Inspection and Quarantine Technology Center of Shandong Entry Exit Inspection and Quarantine Bureau
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Inspection and Quarantine Technology Center of Shandong Entry Exit Inspection and Quarantine Bureau
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Abstract

The invention discloses an identification medium for generating aflatoxin strains and an identification method of the same. The medium comprises the following substances, by weight: 5 to 15 g of potato starch, 15 to 30 g of glucose, 10 to 20 g of agar, 5 to 15 g of methylated beta-cyclodextrin, and 1 L of distilled water. The medium has a pH value between 5 and 6. The method comprises the following steps: (1) preparation of the medium and (2) culture. The step (1) comprises: dissolving the components in the 1 L of distilled water and boiling the mixture, cooling the mixture a little and adjusting the pH value to between 5 and 6, and putting the medium in a culture dish. The step (2) comprises: after the medium solidifies, inoculating to-be-identified strains on the medium, culturing at 28 DEG C for 5 days, and observing the results under a 365 nm ultraviolet lamp. The raw material of the medium can be easily obtained, and the medium is improved in effect and reduced in cost, and thus the medium has great value for applications and promotion. The method achieves easy operation, does not need special instruments or equipment, and is quick, convenient, accurate and reliable.

Description

A kind of evaluation substratum and authentication method thereof of aflatoxigenic strain
Technical field
The present invention relates to substratum and authentication method thereof that a kind of aflatoxigenic strain is identified, belong to microorganism identification culture technique field.
Background technology
Aflatoxin (Aflatoxins) is mainly the mycotoxins that has very strong toxicity and carinogenicity by the class that flavus and Aspergillus parasiticus produce, extensively be present in the agricultural-food such as peanut, corn, wheat class, paddy serious harm people, animal, avian health.All bacterial strains of Aspergillus parasiticus all can produce B group and G group aflatoxin, and flavus only has the part bacterial strain can produce aflatoxin, and can only produce B group aflatoxin.
Study and find, flavus is modal fungi in grain and food, but not all aspergillus flavus strains can produce toxin,, according to the people such as Matles report, from the aspergillus flavus strain that occurring in nature separates, only has 10%-30% energy toxin producing; Produce the fungi of aflatoxin and can infect multiple kinds of crops and growth therein, before harvesting time in farm crop the pollution of aflatoxin mainly due in its growth later stage, hot, dry climate condition occurring, this condition can stimulate the fungal growth that produces aflatoxin, therefore when this weather appears in the grown place big area, will cause farm crop significantly to infect the phenomenon of aflatoxin; , if farm crop meet with sick worm harm, soil depletion, early frost, lodging and moist rainy etc. to the disadvantageous condition of plant growth in process of growth, also can impel the generation of aflatoxin; After results, the pollution of aflatoxin mainly causes because condition of storage is improper, and as high in the storage temperature, high humidity, ventilated condition are bad etc.According to FAO, the whole world is annual approximately has 25% farm crop to suffer the pollution of mould and toxin thereof, 2% farm crop is approximately arranged because of seriously polluted nutrition and the economic worth of losing.Estimate thus, the annual direct or indirect financial loss that causes because of mycotoxin contamination in the whole world reaches tens billion of dollars.China is Peanut big producing country, and peanut ultimate production and export volume all account for the first in the world, is China's foreign exchange earning agricultural-food with obvious competitive edge few in number.In recent years,, due to the peanut aflatoxin problem, affect the hygienic quality of China's peanut, restricted the outlet of China's peanut.
The seriousness of aflatoxin contamination problem is well-known, but at present the focus of research mainly concentrates in detection to aflatoxin, really from bacterial strain, does not produce this source of poison and does not carry out effective prevention and control; Domestic evaluation to Toxigenic fungi at present is mainly according to morphological feature, and for flavus, does not distinguish and produce poison and not toxigenic bacterium strain.
Summary of the invention
Technical problem to be solved by this invention is: provide a kind of formula simple, result aflatoxigenic strain is accurately and reliably identified substratum and is identified the method for aflatoxigenic strain.
For solving the problems of the technologies described above, the technical solution used in the present invention is:
The substratum that a kind of aflatoxigenic strain is identified, is characterized in that, comprises the material of following weight: yam starch 5-15g, glucose 15-30g, agar 10-20g, methylated β-cyclodextrin 5-15g, distilled water 1L; The pH value of described substratum is 5-6.
Preferably, in above-mentioned substratum, the content of various materials is: yam starch 10g, glucose 20g, agar 15g, methylated β-cyclodextrin 8g, distilled water 1L.
The applicant studies discovery, adds (NH 4) 2SO 4And ZnSO 4Can stimulate the toxin of bacterial strain to produce.
Preferably, substratum comprises that the content of above-mentioned two kinds of inorganic salt is: 1-8g (NH 4) 2SO 4And 0.01-0.06gZnSO 4.
Preferably, the amount of above-mentioned two kinds of inorganic salt is: 4g (NH 4) 2SO 4And 0.02gZnSO 4.
Preferably, the amount of above-mentioned two kinds of inorganic salt is: 2g (NH 4) 2SO 4And 0.06gZnSO 4.
A kind of method of identifying aflatoxigenic strain, comprise the following steps:
(1) preparation of substratum: with 5-15g yam starch, 15-30g glucose, 10-20g agar, 5-15g methylated β-cyclodextrin, 1-8g (NH 4) 2SO 4And 0.01-0.06gZnSO 4Adding distil water 1L dissolves and boils, and little cool rear adjusting pH value is 5-6, and substratum is poured in culture dish;
(2) cultivate: after culture medium solidifying, the inoculation of needs evaluation on substratum, was cultivated 5 days observations under the ultraviolet lamp of 365 nm for 28 ℃.
Culture medium raw material of the present invention is easy to get, and improves effect, saves cost, has great application and promotional value; Identify that the method for producing malicious aspergillus flavus strain is simple to operate, do not need special plant and instrument, quick, easy, accurate and reliable.
Description of drawings
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in further detail.
Fig. 1 is the bacterium colony figure of bacterial strain on the described substratum of embodiment 1 that produces aflatoxin.
Fig. 2 is the bacterium colony figure of bacterial strain on common mould medium that produces aflatoxin.
Fig. 3 is the bacterium colony figure of bacterial strain on substratum of the present invention of aspergillus flavus not producing toxin.
Fig. 4 does not produce the bacterium colony figure of malicious aspergillus flavus strain on common mould medium.
Fig. 5 is the bacterium colony comparison diagram of aflatoxigenic strain on embodiment 2 and the described substratum of embodiment 3, and wherein left hand view is the bacterium colony figure of embodiment 2, and right part of flg is the bacterium colony figure of embodiment 3.
Embodiment
Embodiment 1
The substratum that a kind of aflatoxigenic strain is identified, comprise the material of following weight: yam starch 10g, glucose 20g, agar 15g, methylated β-cyclodextrin 8g, distilled water 1L.
The preparation method of above-mentioned substratum is: 10g yam starch, 20g glucose, 15g agar, 8g methylated β-cyclodextrin adding distil water 1L are dissolved and boil, and little cool rear adjusting pH value is 5-6, is poured in culture dish substratum cooling standby.
Embodiment 2
A kind ofly produce the substratum that the aflatoxin trichoderma strain is identified, comprise the material of following weight: yam starch 10g, glucose 20g, agar 15g, methylated β-cyclodextrin 8g, distilled water 1L.
Above-mentioned substratum also comprises the material of following weight: 4g (NH 4) 2SO 4And 0.02gZnSO 4.
The preparation method of this substratum: with 10g yam starch, 20g glucose, 15g agar, 8g methylated β-cyclodextrin, 4g (NH 4) 2SO 4: and 0.02gZnSO 4Adding distil water 1L dissolves and boils, and little cool rear adjusting pH value is 5-6, is poured in culture dish substratum cooling standby.
Embodiment 3
A kind ofly produce the substratum that the aflatoxin trichoderma strain is identified, comprise the material of following weight: yam starch 10g, glucose 20g, agar 15g, methylated β-cyclodextrin 8g, distilled water 1L.
Above-mentioned substratum also comprises the material of following weight: 2g (NH 4) 2SO 4And 0.06gZnSO 4.
The preparation method of this substratum: with 10g yam starch, 20g glucose, 15g agar, 8g methylated β-cyclodextrin, 6g (NH 4) 2SO 4: and 0.06gZnSO 4Adding distil water 1L dissolves and boils, and little cool rear adjusting pH value is 5-6, is poured in culture dish substratum cooling standby.
Embodiment 4
A kind of method of identifying aflatoxigenic strain, comprise the following steps:
(1) preparation of substratum: with 5-15g yam starch, 15-30g glucose, 10-20g agar, 5-15g methylated β-cyclodextrin, 1-8g (NH 4) 2SO 4And 0.01-0.06gZnSO 4Adding distil water 1L dissolves and boils, and little cool rear adjusting pH value is 5-6, and substratum is poured in culture dish;
(2) cultivate: after culture medium solidifying, the inoculation of needs evaluation on substratum, was cultivated 5 days observations under the ultraviolet lamp of 365 nm for 28 ℃.
Embodiment 5 The specificity experiment
Toxigenic fungi common in the several foods has been chosen in experiment, the bacterial strain of flavus, Aspergillus ochraceus, aspergillus versicolor, Aspergillus nidulans, Aspergillus fumigatus, excellent aspergillus, Penicillium citrinum and reaping hook, all experimental strains are seeded in substratum of the present invention, cultivated 5 days observations under the ultraviolet lamp of 365 nm for 28 ℃.Substratum of the present invention in the present embodiment is selected the formula in embodiment 1.
As Figure 1-4, Fig. 1 is the bacterium colony figure of aflatoxigenic strain on substratum of the present invention to result; Fig. 2 is the bacterium colony figure of aflatoxigenic strain on common mould medium; Fig. 3 is the bacterium colony figure of bacterial strain on substratum of the present invention of aspergillus flavus not producing toxin; Fig. 4 is the bacterium colony figure of aspergillus flavus not producing toxin bacterial strain on common mould medium.
Result shows, only have Fig. 1 bluish voilet fluorescence to occur, aflatoxin can produce the basket purple fluorescence by its structures shape under ultraviolet ray, other bacterial strain also produces mycotoxins such as ochratoxin, sterigmatocystin, citrinin, the red ketenes of corn etc., but it does not produce bluish voilet fluorescence, the fluorescence that this substratum can only specific enhancing aflatoxin is described, other toxin is not produced same function.
Embodiment 6 Controlled trial
(1) add or do not add the substratum of inorganic salt
Substratum in this experimental selection the above embodiments 1 and embodiment 2 compares test, test (NH 4) 2SO 4And ZnSO 4On the impact that stimulates the aspergillus flavus strain toxin to produce.Other culture condition are identical.
(2) add the inorganic salt of different concns
Substratum in this experimental selection the above embodiments 2 and embodiment 3 compares test, (the NH of test different concns 4) 2SO 4And ZnSO 4On the impact that stimulates the aspergillus flavus strain toxin to produce.Other culture condition are identical.
The amount of adding in embodiment 2 is 4g (NH 4) 2SO 4And 0.02gZnSO 4The amount of adding in embodiment 3 is 2g (NH 4) 2SO 4And 0.06gZnSO 4.
Fig. 5 is the growth conditions of aflatoxigenic strain on embodiment 2 and embodiment 3 substratum, and wherein left hand view is embodiment 2, and right part of flg is embodiment 3, (NH as seen from Figure 5 4) 2SO 4And ZnSO 4Two kinds of inorganic salt have certain influence to the growth of aflatoxigenic strain, as (NH 4) 2SO 4And ZnSO 4Concentration in suitable scope the time, can stimulate strain growth.
Comparison diagram 1 and Fig. 5, can find out, adds appropriate interpolation (NH in substratum 4) 2SO 4And ZnSO 4Can stimulate the growth of bacterial strain and produce poison, can strengthen the fluorescence intensity of toxin in substratum, and adding the inorganic salt of different concns, the fluorescence intensity of periphery of bacterial colonies is also different.

Claims (6)

1. an aflatoxigenic strain is identified substratum, it is characterized in that, comprises the material of following weight: yam starch 5-15g, glucose 15-30g, agar 10-20g, methylated β-cyclodextrin 5-15g, distilled water 1L; The pH value of described substratum is 5-6.
2. aflatoxigenic strain according to claim 1 is identified substratum, it is characterized in that, comprises the material of following weight: yam starch 10g, glucose 20g, agar 15g, methylated β-cyclodextrin 8g, distilled water 1L; The pH value of described substratum is 5-6.
3. aflatoxigenic strain according to claim 1 and 2 is identified substratum, it is characterized in that, also comprises the material of following weight: 1-8g (NH 4) 2SO 4And 0.01-0.06gZnSO 4.
4. aflatoxigenic strain according to claim 1 and 2 is identified substratum, it is characterized in that, also comprises the material of following weight: 4g (NH 4) 2SO 4And 0.02gZnSO 4.
5. aflatoxigenic strain according to claim 1 and 2 is identified substratum, it is characterized in that, also comprises the material of following weight: 2g (NH 4) 2SO 4And 0.06gZnSO 4.
6. a method of identifying aflatoxigenic strain, is characterized in that, comprises the following steps:
(1) preparation of substratum: with 5-15g yam starch, 15-30g glucose, 10-20g agar, 5-15g methylated β-cyclodextrin, 1-8g (NH 4) 2SO 4And 0.01-0.06gZnSO 4Adding distil water 1L dissolves and boils, and little cool rear adjusting pH value is 5-6, and substratum is poured in culture dish;
(2) cultivate: after culture medium solidifying, the inoculation of needs evaluation on substratum, was cultivated 5 days for 28 ℃, observations under the ultraviolet lamp of 365 nm, what bluish voilet fluorescence occurs can produce the bacterial strain of aflatoxin exactly.
CN2013102962497A 2013-07-16 2013-07-16 Identification medium for generating aflatoxin strains and identification method of the same Pending CN103387941A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106434836A (en) * 2016-08-29 2017-02-22 中国水稻研究所 Method for quickly and efficiently identifying aflatoxin producing strain
CN107557402A (en) * 2014-12-04 2018-01-09 武汉轻工大学 A kind of AFG2 of aspergillus parasiticus produces malicious fermentation process
CN110973163A (en) * 2019-12-18 2020-04-10 吉林大学 Liquid microbial inoculum for biologically preventing, controlling and spraying aflatoxin

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
许艳丽 等: "一种快速检测产黄曲霉毒素菌株的方法", 《食品与发酵工业》 *
许艳丽: "产黄曲霉毒素菌株的检测方法及产毒条件的研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑 E055-21》 *
鲍蕾 等: "液相色谱法检测植物油中黄曲霉毒素B_1、B_2、G_1、G_2", 《山东出入境检验检疫局专刊》 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107557402A (en) * 2014-12-04 2018-01-09 武汉轻工大学 A kind of AFG2 of aspergillus parasiticus produces malicious fermentation process
CN107557402B (en) * 2014-12-04 2019-04-26 武汉轻工大学 A kind of malicious fermentation process of AFG2 production of aspergillus parasiticus
CN106434836A (en) * 2016-08-29 2017-02-22 中国水稻研究所 Method for quickly and efficiently identifying aflatoxin producing strain
CN110973163A (en) * 2019-12-18 2020-04-10 吉林大学 Liquid microbial inoculum for biologically preventing, controlling and spraying aflatoxin
CN110973163B (en) * 2019-12-18 2021-04-09 吉林大学 Liquid microbial inoculum for biologically preventing, controlling and spraying aflatoxin

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Application publication date: 20131113