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From: Food Quality & Safety magazine, February/March 2012

How to Implement a Mycotoxin HACCP System

by Patricia Knass

The use of HACCP systems to guarantee the production of safe food products for consumers has become very popular over the past few decades. The HACCP technique is a logical, straightforward control system based on the prevention of problems; in other words, the HACCP program uses common sense to manage food safety.1-2

To implement a functioning HACCP system, five successive steps are recommended:

  • Observing the process/product from beginning to end;
  • Identifying potential hazards and determining which part of the process they may arise in;
  • Establishing controls and supervising them;
  • Keeping written records of everything; and
  • Ensuring that the system continues to work efficiently.

The object of this article is to present adequate options for monitoring control points in a mycotoxin HACCP system. Of course, a good HACCP system has to be capable of coping with all factors that put the production chain at risk, not just mycotoxins.

First, certain concepts must be made clear, especially regarding the characterization of hazardous mycotoxins. Although mycotoxins are chemical compounds that appear as residues in food, they are considered biological hazards instead of chemical hazards because their presence is a direct consequence of fungal contamination occurring at some point in the system.3-4

Cereals and nuts are the food products most sensitive to mycotoxin contamination. Nonetheless, these residues can also be detected in products of animal origin, such as milk, meat, and eggs, and in plant products such as coffee, wine, and dried fruit, among others. The number of foods that can be contaminated by mycotoxins is as vast as the types of contaminating mycotoxins; therefore, establishing a single model for a mycotoxin control system in foods is no simple task.

Monitoring Methods: Fungal or Mycotoxin Analysis?

The fact that mycotoxins are considered biological hazards could lead us to believe that the correct methods for monitoring control points are those that detect, quantify, identify, and classify fungi. However, we must first consider that although in many cases the different processes eliminate fungi from the substrate, mycotoxins are so stable they remain throughout the food processing chain; in other situations, potential mycotoxigenic fungi might be detected but are not producing toxins because it is either a non-producing strain, the substrate is inadequate, or just because the environmental conditions are not propitious for generating the mycotoxin in question.

In addition to a possible lack of correlation between the presence of potentially toxigenic fungi and mycotoxins in a particular food product, we must consider the available methods of fungal analysis in foods:

  • Counting, identification, and classification: The disadvantage of these characterization methods for detecting microbial contamination is the time required to reach a useful answer—in other words, counting and identifying potentially toxigenic strains and their capacity to produce mycotoxins in the studied substrate. Obtaining the end results for a single isolated strain can take between two to three weeks, highly inconvenient for monitoring a control point in a food production process.
  • Molecular biological methods: Several methods for detecting toxigenic fungi based on molecular biology are being investigated. However, the most approximate methods are those based on real-time polymerase chain reaction, which have a number of limitations when applied at an industrial scale. First, few specific genes involved in the production of mycotoxins have been established—the fusarium genes Tri4, Tri5 and Tri6 for detecting species producing Group A and Group B trichotecenes are an example. Moreover, although genes associated with other species producing aflatoxins and sterigmatocystin (nor-1, ver-1, aflR, omt-A, among others) are useful for discriminating these species from other fungi, they are not highly specific and it is not clear whether they can discern mycotoxigenic strains from non-producing strains within a particular species—for example aspergillus flavus. Another disadvantage of using real time PCR for detecting toxigenic fungi is that it is only capable of detecting the fungi when it is actively producing toxins, limiting the use of this test when the fungi are inactive or growing under conditions that are unfavorable for producing mycotoxins.
  • The future of detecting and quantifying mycotoxigenic fungi is based on microarray technology (biochips). This technique allows comparative and simultaneous analyses of hundreds of genes in a device similar to a slide, in a very short period of time. However, many studies have yet to be undertaken that include species-specific gene fragments and sequences with phylogenetic information on the potential mycotoxin-producing species, together with essential genes for mycotoxin biosynthesis.5-6

The previous description not only shows how highly complex and time-consuming it is to establish a monitoring system at a control point by trying to determine toxigenic fungi, but it also highlights the limitations of the methods used for detecting whether the fungi is producing the mycotoxins we are trying to control. Hence, the aforementioned methods are very useful for establishing studies regarding the existence and production of mycotoxins by fungi in certain foods and environments, which would allow a hazard analysis, but are not applicable in the monitoring HACCP system once implemented. For this reason, methods that analyze the mycotoxins present along the production chain of food products are chosen for monitoring control points.

The analytical method selected depends on a number of factors such as the type of sample, the levels of mycotoxins to be detected, the control point to be monitored, and the availability of technological, economic, and human resources to perform the determination.7

The fastest methods used in the food industry are those based on enzyme-linked immunosorbent assay technology, using wells that provide quantitative results of many products at a reasonable cost. It is important to take into account the fact that the individual test kit must be validated for the mycotoxin and commodity in question. Therefore, when choosing a determined ELISA test kit it is important to confirm that it is applicable to the product to be analyzed. Many times, these types of determinations, which take between 10 and 20 minutes, are used by industries for monitoring all the control points, even those requiring an analytical reference system.8-11

There are other commercially available methods for detecting mycotoxins that provide even faster determination, and these are especially useful at the reception point of raw materials. These are also immunological methods, like ELISA, but are performed using a strip-type format that visually indicates the presence or absence of a particular mycotoxin using a reference cut-off point. Alternatively, the strip is read with a specific reader that is able to supply the user with a quantitative test result.

Biosensors are being evaluated for use with liquid products such as wine, milk, and beer, and these will have the advantage of online results, ensuring permanent control point monitoring. The systems that have been developed so far require extracting the mycotoxin from the substrate with a solvent, but methods that can use the liquid matrix itself are under investigation.12-13

INSERT_CAPTION
The fungus aspergillus flavus: Mycotoxigenic or not?

Confirmation and Certification

The quality management plan must consider the correct functioning of the monitoring methods. There are two ways to ensure this, and because they complement each other, both options can be used to verify that the procedures provide precise and exact results.

The first option involves using matrix reference materials or internal control samples. These are product samples used to audit the analytical procedure, because they contain a known level of contamination with the mycotoxin of interest and their certificates include the value of uncertainty of determining the mycotoxin in the product. This reference or control sample is tested in the analytical system used, at a frequency established according to the number of analyses performed at that point.

The other option is to send samples from the different monitoring points to contract laboratories with analytical systems that can certify the value of the samples. This is also performed to confirm results that are very close to the acceptance or rejection point of the critical limit established. These laboratories use chromatographic techniques that are highly sensitive and precise, such as HPLC, TLC, LC-MS/MS, and GC-ECD. If possible, the laboratory must also be a certified or accredited entity (e.g., ISO 17025) or have a good quality assurance plan to guarantee accurate results that are independent of the equipment or professionals employed.15-16

Throughout this article, compliance with the premises described at the beginning has been outlined. You must understand that the chemical compound being analyzed is characterized as a biological hazard because it is related to the presence of a mycotoxigenic fungus.

Monitoring mycotoxins throughout the production chain is one safety measure. Other tools that can be used to prevent contamination include monitoring for environmental conditions that might favor the production of mycotoxigenic fungi, by measuring water activity and temperature in exposed areas.

 


Patricia Knass studied biochemistry at the National University of Misiones in Argentina and earned her master’s in food technology at the University of Buenos Aires. Since 2006, she has served as a technical adviser to Romer Labs. She is also the co-founder and CEO of AgriNEA, a consultancy company with offices in Argentina and Paraguay that focuses on food safety and agriculture.

 

References

  1. Mortimore S, Wallace C. HACCP: A Practical Approach. 2nd ed. Gaithersburg, Md.: Aspen Publishers, Inc.; 1998.
  2. American Society for Quality: Food, Drug, and Cosmetics Division. The Quality Auditor’s HACCP Handbook. Milwaukee, Wis.: ASQ Quality Press; 2001.
  3. FAO/IAEA Training and Reference Centre for Food and Pesticide Control. Manual on the application of the HACCP system in mycotoxin prevention and control. Food and Agriculture Organization of the United Nations website. 2001. Reprinted 2003. Available at: www.fao.org/docrep/005/y1390e/ y1390e00.htm. Accessed Jan. 16, 2012.
  4. Aldred D, Magan N, Olsen M. The use of HACCP in the control of mycotoxins: the case of cereals. In: Magan N, Olsen M, eds. Mycotoxins in Food: Detection and Control. Cambridge, England: Woodhead Publishing Ltd; 2004.
  5. Nicholson P. Rapid detection of mycotoxigenic fungi in plants. In: Magan N, Olsen M, eds. Mycotoxins in Food: Detection and Control. Cambridge, England: Woodhead Publishing Ltd; 2004.
  6. Cabañes FJ, Abarca ML, Bragulat MR, Castellá G. Especies productoras de micotoxinas. In: Del Castillo JMS. Micotoxinas en Alimentos. Madrid: Díaz de Santos Editorial; 2007.
  7. Knass PS. Micotoxinas en la industria alimentaria: mantener la situación bajo control. Énfasis Alimentación online. Available at: www.alimentacion.enfasis.com/contenidos/nota.html?idNota=7575. Accessed Jan. 18, 2012.
  8. Zheng Z, Humphrey C, King RS, Richard JL. Validation of an ELISA test kit for the detection of total aflatoxins in grain and grain products by comparison with HPLC. Mycopathologia. 2005;159(2):255-263.
  9. Zheng Z, Hanneken J, Houchins D, King RS, Lee P, Richard JL. Validation of an ELISA test kit for the detection of Ochratoxin A in several foods commodities by comparison with HPLC. Mycopathologia. 2005;159(2):265-272.
  10. Zheng Z, Richard JL, Binder H. A review of rapid methods for the analysis of mycotoxins. Mycopathologia. 2006;161(5):261-273.
  11. Zheng ZM. Validation report of AgraStrip Aflatoxin Test, Romer Labs. Study number SIN-2005- 01. 2005.
  12. Ngundi MM, Shriver-Lake LC, Moore MH, Lassman ME, Ligler FS, Taitt CR. Array biosensor for detection of ochratoxin A in cereals and beverages. Anal Chem. 2005;77(1):148-154.
  13. Van der Gaag B, Spath S, Dietrich H, et al. Biosensors and multiple mycotoxin analysis. Food Control. 2003;14(4):251-254.
  14. Häubl G, Berthiller F, Krska R, Schuhmacher R. Suitability of a fully 13C isotope labeled internal standard for the determination of the mycotoxin deoxynivalenol by LC-MS/MS without clean up. Anal Bioanal Chem. 2006;384(3):692-696.
  15. Barendsz AW. Food safety and total quality management. Food Control. 1998;9(2):163-170.
  16. Berg T. How to establish international limits for mycotoxins in food and feed? Food Control. 2003;14(12):219-224.

 

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