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Enumeration of yeasts and moulds and production of toxins
Philip B. Mislivec and Michael E. Stack
The large and diverse group of microscopic foodborne yeasts and moulds (fungi) includes several hundred species. The ability of these organisms to attack many foods is due in large part to their relatively versatile, environmental requirements. Although all yeasts and moulds are obligate aerobes (require free oxygen for growth), their acid/alkaline requirement for growth is quite broad, ranging from pH 2 to above pH 9. Their temperature range (10-35°C) is also broad, with a few species capable of growth below or above this range. Moisture requirements of foodborne moulds are relatively low; most species grow at a water activity (aw) of 0.85 or less, although yeasts generally require a higher water activity.
Both yeasts and moulds cause various degrees of deterioration and decomposition of foods. They can invade and grow on virtually any type of food at any time, e.g., they invade field crops such as small grains, nuts, beans, tomatoes, and apples both in the field before harvesting and during storage. They also grow on processed foods and food mixtures. Their detectability in or on foods depends on food type, organisms involved, and degree of invasion, i.e., the contaminated food may be slightly blemished, severely blemished, or completely decomposed, with the actual yeast or mould growth manifested by rot spots of various sizes and colors, unslightly scabs, slime, white cottony mycelium, or highly colored sporulating mould. Abnormal flavors and odors may also be produced. Occasionally a food appears to be mould-free but upon mycological examination, is found to be contaminated. Contamination of foods by yeasts and moulds can also result in substantial economic losses to producer, processor, and consumer.
Several foodborne moulds, and possibly yeasts, may also be a potential hazard to human or animal health because of their ability to produce toxic metabolities known as mycotoxins. Most mycotoxins are stable compounds that are not destroyed during food processing or home cooking. Even though the generating organisms may not survive food preparation, the preformed toxin may still be present. Certain foodborne moulds and yeasts may also be a hazard because of their ability to elicit allergic reactions or even cause infection. Although most foodborne fungi are not infectious, some species can cause infection, especially to vulnerable population groups, e.g., the aged and debilitated and individuals who are receiving chemotherapy or antibiotic treatment.
The dilution plating and the direct plating methods may be used to detect fungi in foods. The dilution plating method, which is the traditional method used in examining foods, is given here. It varies only slightly from the method described in previous editions of the Bacteriological Analytical Manual. The direct plating method is included here because we have found it to be more efficient than the dilution plating method for detecting individual mould species, including most of the toxin producers. (Note: It is less effective in detecting yeasts.) It can also be used to determine whether the presence of mould is due to external contamination or internal invasion. Methodology for testing the ability of isolates of toxigenic mould species to produce mycotoxins on sterile rice water substrate is also included.
Enumeration of Yeasts and Moulds in Foods-Dilution Plating Technique
A. Equipment and materials
B. Media and reagents
C. Analysis of samples
- Antibiotics. Use of antibiotics is preferred to tartaric acid solution because stock solutions are relatively easy to prepare yeast and mould species, does not result. Chiortetracycline-HCl, at agar medium concentration of 40 ppm, is recommended. Other antibiotics may be used (e.g., chloramphenicol, streptomycin) but should always be used at the same concentration as chlortetracycline-HCl and in addition to it. Prepare stock solutions by dissolving 1 9 of antibiotic in 100 ml of sterile distilled water and filtering through a 0.45 µm membrane (Nalge Sybron Corp., Rochester, NY). Store stock solutions in dark at 4-8°C. Shelf life should exceed 1 month. Equilibrate stock solutions to room temperature immediately before use. If agar medium is in 250 ml aliquots, add 1 ml of 100 ml stock solution to obtain 40 ppm concentration. If medium aliquots are greater or less, adjustments will be necessary.
- Tartaric acid solution. A 10% solution may be used to adjust agar medium to pH 3 5 ± 0.1 Sterilize solution by filtering through 0.45 µm membrane. Titrate to determine amount of solution needed to adjust pH to 3.5. Type and aliquot volume of medium will affect amount of solution needed. After adding solution to medium, verify pH by letting a portion of medium solidify and checking with pH meter. Do this for every new lot of medium prepared.
ENUMERATION OF MOULDS IN FOODS-DIRECT PLATING TECHNIQUE FOR FOODS (SUCH AS DRIED BEANS, NUTS, WHOLE SPICES, COFFEE AND COCOA BEANS) THAT CAN BE HANDLED WITH FORCEPS
A. Equipment and materials
B. Media and reagents
C. Analysis of non-surface-disinfected (NSD) foods
D. Analysis of surface-disinfected (SD) foods
Perform disinfection in clean laboratory sink, not stainless steel, free from any acid residues, with tap water running (precautions against chlorine gas generation). Using rubber gloves, transfer about 50 9 of sample into sterile 150 ml beaker. Cover with 5% NaOCI solution for 1 min. swirling beaker contents gently but constantly in clockwisecounterclockwise motion Decant 5% NaOCI solution and give beaker contents three 1 min sterile distilled water rinses. Prepare plates, plate sample, incubate, and read plates as in C, 2-5, above. Comparison of NSD and SD results from same sample will indicate whether mouldiness was due mainly to surface contamination or to internal invasion and growth.
METHODS FOR DETERMINING TOXIN PRODUCTION BY MOULDS
A. Equipment and materials
B. Media and reagents
C. Toxin production
Into 300 ml wide-mouth Erlenmeyer flask, add 50 9 rice and 50 ml distilled water. Cotton plug flasks and autoclave 20 min at 121°C and 15 psi. Aseptically multispore-inoculate separate cooled flasks with individual mould isolates. Incubate inoculated flasks at 22-25°C until entire surface is covered with growth and mycelium has penetrated to bottom of flask (15-20 days). To each flask, add 150 ml chloroform (150 ml methanol if toxin in question is deoxynivalenol), using short-stem glass funnel inserted alongside unremoved cotton plug (to minimize mould spore dissemination). Hear flask contents in fume hood on steam bath until solvent begins to boil. (Conduct all subsequent steps in fume hood.) With spatula, break up mouldy rice cake and transfer flask contents into explosionproof blender and blend at high speed for 1 min. Filter blender contents through filter paper inserted into short-stem glass funnel. Collect filtrate in 300 ml Erlenmeyer flask. Return rice cakes to blender, add 100 ml unheated solvent and blend 1 min at high speed. Filter as above and combine filtrates. Add boiling chips to flask containing filtrates and steam evaporate to 20-25 ml. If analysis is not to follow immediately, evaporate to dryness and store flask in dark. Rinse all glassware, etc., used for extraction in 5% NaOCI solution before soap and water cleansing. Rice cake should be submerged in 5% NaOCI solution for 72 h before autoclaving and disposal.
D. Toxin analysis
Toxin analysis requires use of appropriate mycotoxin standards for both qualitative and quantitative evaluation. Use either thin layer chromatography or high performance liquid chromatography, as described in Chapter 26, of Official Methods of Analysis 14th ea., 1984 (Association of Official Analytical Chemists, Arlington, VA), for determination of most foodborne mycotoxins.