ABSTRACT
INTRODUCTION Fungal spoilage is a cause of immense food losses during storage of crops and food products. In order to overcome food spoilage by fungi, mankind has developed an array of methods to make food, which can be regarded as a rich and complex medium, difficult to colonize. This is done, among others, by means of lowering of the water activity by addition of sugars, salt or after drying. In other cases, food products are kept under lowered temperatures. A third possibility is the killing of fungi present in the food matrix by a transient heat treatment (pasteurisation). Currently, novel techniques are introduced as high-pressure treatment or storage of a food product under a modified atmosphere. Although heat treatment is a reliable and historical method, a class of fungal organisms is able to survive prolonged periods of heat and these organisms cause a restricted, but consistent, loss of food products. This is especially interesting with modern trends to use minimally processed food where the heat treatment is minimalised in favour of the organoleptic properties of the product and vitamin content. HEAT RESISTANCE IN FUNGI Vegetative (yeast) cells usually have little heat resistance. When heated in beer (pH 4.0, 5% ethanol) Saccharomyces carlsbergensis, Hansenula anomala and Pichia membranaefaciens had D49-values of less than 2 min. S. willianus, was
more heat-resistant with a D49 around 15 min (Tsang and Ingledew, 1982). For Zygosaccharomyces bailii, Saccharomyces bisporus, H. anomala and Z. rouxii decimal reduction was observed after heating for 1 min in a buffer (pH 5,5) supplied with 400 g/l sucrose, at 56, 55, 54 and 50°C, respectively (Baggerman and Samson, 1988). Yeast species that were heated in grapefruit serum showed similar characteristics (Parish, 1991). The presence of sucrose, sodium chloride or glycerol in the heating menstruum was somewhat protective during heating, while sorbate and benzoate usually reduced the heat resistance of the yeast cells (Beuchat, 1981; Agab and Collins, 1992). Living (and growing) fungal cells exhibit after a sudden increase in temperature of approximately 10 °C above the optimal growth temperature, a phenomenon called heat shock. Increases in temperature lead to a number of changes including protein denaturation, cell cycle arrest and changes in the fluidity of the membrane. One of the most deleterious factors in heat shocked cells is the disturbance (unfolding) of the protein structure that leads to the formation of aggregates of proteins that affect the functioning of the cell lethally (Riezman, 2004). Accumulation of compatible solutes (glycerol, trehalose, mannitol) that protect the cellular membrane and the proteins (Crowe et al., 1984; Prestrelski et al., 1993) is one of the answers of the cell to heat shock. In addition, heat shock proteins play an important role in cooperation with compatible solutes in yeasts (Elliott et al., 1996), but these are scarcely studied in fungal survival structures. Recently, the genome of the
filamentous fungus Aspergillus fumigatus was sequenced (Nierman et al., 2005) and 323 genes showed higher expression at 48 °C compared to 37 °C. The most strongly upregulated genes included three proteins related to compatible solute synthesis and degradation and nine heat shock proteins. This also illustrates the role of different factors during the heat-shock response. Fungal survival structures as conidia, sclerotia, chlamydospores and ascospores can be regarded as more or less heat resistant when they are compared to actively growing fungal cells. They do not exhibit the metabolism of the vegetative cells, but prevail in a dormant state and often are encompassed by a thicker cell wall. Conidia are non-motile asexual spores of many fungal species that are often dispersed by air and water and which exhibit some dormancy, namely that they germinate only when proper nutrients are present in the medium. Conidia of the fungal species Aspergillus niger, Penicillium chrysogenum, Wallemia sebi, Eurotium rubrum and P. glabrum, exhibit a decimal reduction in buffer in the range of 56 and 62 °C (Baggerman and Samson, 1988) and P. roqueforti, P. expansum, P. citrinum and A. flavus have D-values between 3.5 and 230 min at 54-56 °C (Bröker et al., 1987a). The age of the spores at the time of harvest as well as the composition of the growing and heating media influence the heat resistance. Furthermore, conidia could restore from a heat treatment while P. expansum showed recovery during a 3-day storage in aerated water at 23 °C after a heat treatment at 54 °C compared to conidia that were plated out directly (Baldy et al., 1970). Numbers of colonies were 20-fold higher after this treatment, indicating that the situation after heating has an effect on the number of survivors. Ascospores are widely formed sexual spores within the Ascomycetes with often a high survival capability. In an investigation of 20 yeast strains from soft drinks and fruit products, mainly Saccharomyces cerevisiae, S. bailii (now Z. bailii) and S. chevalieri strains, the D60-values of ascospores were 25 to 350 times
higher than those of the corresponding vegetative cells (Put and de Jong, 1980). In a pH 4,5 buffer S. cerevisiae, S. chevalieri and S. bailii ascospores exhibited D60-values of 22.5, 13 and 10 min (Put and de Jong, 1982). In general, ascospores of filamentous fungi are more heat resistant than mycelia and conidia and more resistant than yeast ascospores. A number of observations suggest that thick-walled hyphal fragments and chlamydospores (Paecilomyces variotii, Fusarium spp) and sclerotia (e.g., in Eupenicillium) can perform high thermo-resistance (Splittstoesser and King, 1984). But by far the most heatresistant fungal structures known to date are ascospores produced by some members of the genera Byssochlamys, Neosartorya and Talaromyces. Ascospores of these fungi are extraordinary heat resistant and show D90 values of several minutes (Beuchat, 1986). In fact, these spores belong to the most resilient eukaryotic structures observed hitherto. A decimal reduction time of 1.5-11 min is observed at 90 °C among different species (Scholte et al., 2001, Dijksterhuis and Samson, 2006). Ascospores of the fungus Talaromyces macrosporus are able to survive at 85 °C for 100 min, which makes these fungal spores as resilient as some bacterial spores (e.g., Bacillus subtilis). One has to realise that heating of cells in a low moisture environment is much less effective. Even yeast cells show very high inactivation times at low humidities. When heated on aluminium foil at a relative humidity of 33-38%, P. membranaefaciens and Rhodotorula rubra cells showed little survival after 5 min at 110 °C (Scott and Bernard, 1985). With D110-values of 1.3, 1.8, 2.9 and 3.6 min, respectively, Debaryomyces hansenii, Kloeckera apiculata, Lodderomyces elongisporus and H. anomala cells were more resistant. HEAT-RESISTANT FUNGI Heat-resistant fungi can survive pasteurising heat treatments of especially high-acid food products (e.g., fruits, see Silva and Gibbs, 2004). Subsequent germination causes spoilage
of canned and pasteurised fruit products. Fungi that are associated with product recalls that cause a damage of millions of dollars in the fruit-juice branch are Byssochlamys nivea (fulva), Talaromyces flavus (macrosporus), Neosartorya fischeri, Eupenicillium brefeldianum (as reviewed by Tournas, 1994). Despite several decades of research these fungi still represent problems in the food branch. Heatresistant fungi are basically soil-borne fungi and fruits that develop in contact with soil (like strawberries and pineapple) are more prone to contamination. The fungus Talaromyces flavus is found to have a worldwide distribution and was isolated from soil samples from 16 different countries including Bermuda, Tasmania, Pakistan and Finland (Fravel and Adams, 1986). The first description of the heat-resistant nature of these fungi in literature was the isolation of Byssochlamys nivea from processed fruit by Olliver and Rendle in the 1930s (Olliver and Rendle, 1934). In 1963, a heatresistant Aspergillus (teleomorph, Neosartorya fischeri) was isolated from canned strawberries (Kavanagh et al., 1963) and later two heatresistant Penicillium-like species were isolated from flash-pasteurised apple juice (van der Spuy et al., 1975) which became later known as Talaromyces (flavus and macrosporus). In addition, other fungal species are now identified as heat-resistant (e.g., Talaromyces trachyspermus, Enigl et al., 1993; Talaromyces helicus and stipitatus, Dijksterhuis and Samson, 2006; Byssochlamys spectabilis, J. Houbraken, unpublished results). INACTIVATION OF HEAT-RESISTANT FUNGI Methods Heat resistance of cells is expressed by means of the D-and Z-values, as is well documented, but the exact measurement and calculation of these values is not so self-evident. Throughout different studies various attempts are made to heat ascospore solutions instantaneously, which is important for an accurate D-value acquisition. These attempts include small
sealed vessels that are plunged into water or oil baths (King and Whitehand, 1990), or a two-phase slug flow heat exchanger (King, 1997) or spiral steel capillary tubes (Engel and Teuber, 1991). A simpler method is the addition of a small aliquots of ascospore suspension to a larger preheated volume of fluid. Dijksterhuis et al. (2002) used small diameter Teflon tubing for sampling of the heated solution without opening the water bath. The D-value, the decimal reduction time, is the time (usually in min) that is needed to inactivate 90% of the microorganisms at a given temperature. Ideally, a linear curve will be obtained when the number of surviving microorganisms, on a logarithmic scale, is plotted against time. However, many cases of non-linear death-rate kinetics are observed (Put and de Jong, 1982; Splittstoesser and King, 1984, King and Halbrook, 1987). King et al. (1979) have provided a mathematical method for these situations. They use the formula: (logN0 – log Nt)α = kt + C, where α is a term that addresses the non-linearity of the death kinetics, and k equals a rate constant and 1/k is a measure for the D-value at the used temperature. Apart from non-linear survivor plots, tailing is a phenomenon where a small subpopulation of spores seems to be extra resistant to heat (Bayne and Michener, 1979; Casella et al., 1990). One cause of tailing can be that a small subpopulation escapes proper heating. Fujikawa and Itoh (1996) modelled the non-linear thermal inactivation of A. niger conidia at 60 °C that included a shoulder, an accelerated decline and a tail. A later report of Fujikawa et al. (2000) indicated that the conidia that adhered to the inner wall of test tubes were the cause of tailing in a test tube system. To compare heat-inactivation rates at different temperatures, a second parameter, the z-value, is used. It denotes the increase or decrease in temperature needed, respectively, to decrease or to increase the D-value by a factor of 10. For A. niger conidia for example, a D59 of 3.3 min with a z-value of 4.9 °C implies a D54 of approximately 33 min (Baggerman and Samson, 1988). Homogeneity of the ascospore suspension is important for heat-resistance measurements;
Table 1. Heat resistance of ascospores at different temperatures and medium composition Fungal species T D-value Medium Reference
Byssochlamys fulva 86° 13–14 Grape juice 1 8 Tomato juice 2 B. nivea 85° 1.3–4.5 Buffer pH 3.5 3 34.6 15° Brix Strawberry pulp 4 88° 8–9 sec Ringer solution 5 90° 1.5 Tomato juice 2 B. spectabilis 85° ca. 70 Buffer, pH 6,8 6 Eurotium herbariorum 70° 1.1–4.6 Grape juice, 65° Brix 7 Eupenicillium
javanicum 85° 3.7 15° Brix strawberry pulp 4
Monascus ruber 80°
1.7–2.0 0.9–1.0
Buffers (pH 3.0 ; pH 7.0) In brine
Neosartorya fischeri 85° 13.2 Apple juice 9 10.1 Grape juice 9 10–60 In ACES-buffer, 10 mM, pH 6.8 10 10.4 Buffer pH 7.0 9 14.5 15° Brix strawberry pulp 4 15.1 15° Brix apple juice 11 19.6–29.5 Dionized water, pineapple juice and concentrate 12 35.3 Buffer pH 7.0 13 88° 1.4 Apple juice 14 4.2–16.2 Heated fruit fillings 15 12.4–17.0 Dionized water, pineapple juice and concentrate 12 90° 4.4–6.6 Tomato juice 2 91° <2 Heated fruit fillings 15 N. pseudofischeri 95° 20 sec 6 Talaromyces flavus 85° 3.3 15° Brix strawberry pulp 4 (macrosporus) 85° 39 Buffer pH 5.0, glucose, 16° 16 20–26 Buffer pH 5.0, glucose 17 88° 7.8 Apple juice 14 7.1–22.3 Heated fruit fillings 15 90° 2-8 Buffer pH 5.0, glucose 17 6.2 Buffer pH 5.0, glucose 9 6.0 Buffer pH 5.0, glucose. Slug flow heat exchanger 9 2.7–4.1 Organic acids 18 2.5–11.1 Sugar content (0-60° Brix) 18 5.2–7.1 PH 3.6-6.6 18 91° 2.1–11.7 Heated fruit fillings 15 T. helicus 70° Ca. 20 19 T. macrosporus 85° 30–100 In ACES-buffer, 10 mM, pH 6.8 20 T. stipitatus 72° Ca. 85 19 T. trachyspermus 85° 45 sec 16 Xeromyces bisporus 82.2° 2.3 21
References: 1. Michener and King (1974). 2. Kotzekidou (1997). 3. Aragão (1989). 4. Casella et al. (1990). 5. Engel and Tueber (1991). 6. J. Houbraken, unpublished data. 7. Splittstoesser et al. (1989). 8. Panagou et al. (2002). 9. Conner and Beuchat (1987b). 10. J. Dijksterhuis, unpublished data, strain CBS 133.64. 11. Gumerato (1995). 12. Tournas and Traxler (1994). 13. Rajashekhara et al. (1996). 14. Scott and Bernard (1987). 15. Beuchat (1986). 16. King (1997). 17. King and Halbrook (1987). 18. King and Whitehand (1990). 19. J. Eleveld, unpublished data. 20. Dijksterhuis and Teunissen (2004). 21. Pitt and Hocking (1982). This is a revised table from: Dijksterhuis, J., and Samson, R. A. (2006). Activation of ascospores by novel food preservation techniques. In Advances in Food Mycology (Hocking, A. D., Pitt, J. I., Samson, R. A., and Thrane, U., eds.). Advances in Experimental Medicine and Biology 571:247-260. Table 1, with kind permission by Springer Science and Business Media.
