H

Fig. 2. Chemical structure of zearalenone.

The Association of Official Agricultural Chemists (AOAC) checks analytical methods through systematic interlaboratory studies to determine the performance characteristics for the intended analytical application; the method is then approved that has a good performance for each matrix (7). In this chapter, we describe some methods to determine AFBj and ZEA in different culture media by high-performance liquid chromatography (HPLC) that are not included by the AOAC, but we usually use them in our laboratory to study the interaction of mycotoxin (or fungal mycotoxin producers) and lactic acid bacteria or adsorbents.

2. Materials

2.1. Growth Media

1. LAPTg broth (8): 15 g/L peptone, 10 g/L triptone, 10 g/L yeast extract, 10 g/L glucose, 0.1% Tween-80, pH 6.5. Sterilize by autoclaving at 121°C for 15 min.

2.2. Reagents

1. Standard solutions of AFBj or ZEA (Sigma, St. Louis, MO). Caution: Perform manipulations under a hood whenever possible, and take particular precautions, such as the use of the glove box, when toxins are in a dry form because of their electrostatic nature and the resulting tendency to disperse in working areas (7).

2. Phosphate-buffered saline (PBS): 16.3 mL 0.5 M KH2PO4, 27.9 mL 0.5 M Na2HPO4, 955.8 mL demineralized water, pH 7.3.

3. Citrate buffer: 46.5 mL 0.1 M citric acid, 3.5 mL 0.1 M sodium citrate, 50 mL demineral-ized water, pH 3.0.

4. Solvents: dimethyl sulfoxide (DMSO) GC, chloroform (reagent grade), benzene (reagent grade), HPLC grade methanol, HPLC grade acetonitrile, HPLC grade water.

2.3. Apparatus

1. Liquid chromatograph: equipped with injection valve and 25-50 ^L loop.

2. Liquid chromatographic column: Reverse phase C18 (5-10 ^m) column, 250 (or 150) x 4.6 mm, with appropriate guard column.

3. Detector: UV detector with variable wavelength or fluorescence detector.

3. Methods

3.1. ZEA in PBS or Citrate Buffer

This is a simple technique; no extraction of ZEA from samples of PBS or citrate buffer is required. Neither of the buffers interferes with the elution of ZEA in the chromatogram.

1. Prepare stock solutions of ZEA in DMSO or methanol.

2. Evaporate the methanol with nitrogen or keep the DMSO.

3. Add PBS, pH 7.3, or citrate buffer, pH 3.0, to achieve the desired volume.

4. Inject directly into the HPLC apparatus (see Note 1).

5. Calculate the relative or absolute concentration of the toxin (see Note 2).

As in Subheading 3.1., no extraction of AFBj from samples of PBS is required. The PBS does not interfere with the elution of this toxin in the chromatogram.

1. Prepare stock solutions of AFBj in benzene/acetonitrile (97:3).

2. Evaporate the benzene/acetonitrile by heating in a water bath at 80°C for 10 min or with nitrogen (see Note 3).

3. Add PBS, pH 7.3, to achieve the desired volume.

4. Inject directly into the HPLC apparatus (see Notes 4 and 5).

5. Calculate the relative or absolute concentration of the toxin (see Note 2).

3.3. ZEA in LAPTg Broth

In some cases, it is necessary to take mycotoxin from a culture medium. Here we describe the method we use in our laboratory for LAPTg broth, which is a general medium used for the growth of lactic acid bacteria. This method is based on the very good solubility of ZEA in chloroform. It could be adapted to AFB1 in other media like LTB broth (9), YES broth (10), or malt extract agar (11).

1. Prepare stock solutions of ZEA in DMSO or methanol.

2. Evaporate the methanol with nitrogen or keep the DMSO.

3. Add LAPTg broth, pH 6.5, to achieve the desire volume.

4. Extract ZEA with two portion of chloroform (3:1 v/v) (see Note 6).

5. Concentrate the chloroform extract to dryness under nitrogen (and by heating in a water bath at 60-65°C).

6. Dissolve in 1 mL of the mobile phase for ZEA.

7. Inject into the HPLC appartus (see Note 1).

8. Calculate the relative or absolute concentration of the toxin (see Note 2).

4. Notes

1. Water/acetonitrile/methanol (1.0:1.6:1.0, v/v/v) is used as the mobile phase with a flow rate of 1 mL/min. Detection is performed by fluorescence with excitation and emission wavelengths of 280 and 440 nm (12), respectively, or 254 nm with a UV detector. The assay is carried out at room temperature with an injection volume of 50 ^L. The retention time is approx 7 min. Before the peak of the toxin, the solvent peak can be observed at 3 min.

2. Toxins are quantified by correlating peak areas of samples with those of standard curves in studies of mycotoxin production by fungi, or by the disappearance of mycotoxin.

3. Methanol at 50 ^L of can be added after evaporating the benzene/acetonitrile; then make it to a desirable volume with PBS.

4. Water/acetonitrile/methanol (6:3:1, v/v/v) is used as the mobile phase with a flow rate of 1 mL/min. Detection is performed by fluorescence with excitation and emission wavelengths of 365 and 418 nm (13), respectively, or 365 nm with a UV detector. The assay is carried out at room temperature with an injection volume of 50 ^L. The retention time is approx 11 min. Before the peak of the toxin, the solvent peak can be observed at 3 min.

5. An aliquot (200 ^L) can be derivatized with trifluoroacetic acid/acetic acid/water (2:1:7) to increase the sensibility of the detection limit. The derivatization solution should be mixed with the aqueous aliquot and kept at 65°C for at least 8.5 min. The derivatized aflatoxin 50 ^L is analyzed using an HPLC/fluorescence detection system with the same mobile phase details as above (precolumn derivatization).

6. The relation of the chloroform/aqueous phase (3:1; v/v) can be indicated for small volumes of aqueous phase (until 10 mL). A relation of 1:1 v/v or less could be fitted for bigger volumes.

References

1. Bueno, D. J., Salvano, M., Silva, J. O., González, S. N., and Oliver, G. (2001) Micotoxinas: diagnóstico y prevención en aves de corral. Bol. Mico. 16, 23-36.

2. Pitt, J. I. (2000) Toxigenic fungi: which are important? Med. Mycol. 1, 17-22.

3. Henry, S., Bosch, F. X., Bowers, J. C., Portier, C. J., Petersen, B. J., and Barraj, L. (1998) Aflatoxins. WHO Food Additives Series 40.

4. Pitt, J. I. and Hocking, A. (eds.) (1997) Fungi and Food Spoilage. Blackie Academic and Professional, London.

5 Trucksess M. W. (2001) Mycotoxins. J. AOAC Int. 84, 202-212.

6. Pitt, J. I., Basílico, J. C., Abarca, M. L., and López, C. (2000) Mycotoxins and toxigenic fungi. Med. Mycol. 38 SI, 41-46.

7. Scott P. M. (1995) Natural Toxins. Official Methods of Analysis of AOAC International, vol, II (Cunniff, P., ed.). AOAC, Arlington, VA, pp. 1-51.

8. Raibaud, P., Galpin, J. V., Ducluzeau, R., Mocquot, G., and Oliver, G. (1973) Le genre Lactobacillus dans le tube digestif du rat I.—Caractères des souches homofermentaires isolées de rats holo et gnotoxéniques. Ann. Microbiol. 124, 83-109.

9 Coallier-Ascah, J. and Idziak, E. S. (1985) Interaction between Streptococcus lactis and Aspergillus flavus on production of aflatoxin. Appl. Environ. Microbiol. 49, 163-167.

10 Davis, N. D., Diener, U. L., and Eldridge, D. W. (1966) Production of aflatoxins B1 and G1 by Aspergillus flavus in a semisynthetic medium. Appl. Microbiol. 14, 378-380.

11. Geisen, R. (1996) Multiplex polymerase chain reaction for the detection of potential aflatoxin and sterigmatocystin producing fungi. System. Appl. Microbiol. 19, 388-392.

12 El-Nezami, H., Polychronaki, N., Salminen, S. and Mykkanen, H. (2002) Binding rather than metabolism may explain the interaction of two food-grade Lactobacillus strains with zearalenone and its derivative a-zearalenol. Appl. Environ. Microbiol. 68, 3545-3549.

13. El-Nezami, H., Kankaanpaa, P., Salminen, S. and Ahokas, J. (1998). Ability of dairy strains of lactic acid bacteria to bind a common food carcinogen, aflatoxin Bj. Food Chem. Toxicol. 36, 321-326.

Other Pathogens

Was this article helpful?

0 0

Post a comment