International Journal of Food Microbiology 95 (2004) 79 88 www.elsevier.

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Prediction of fungal growth and ochratoxin A production by Aspergillus ochraceus on irradiated barley grain as influenced by temperature and water activity
n, V. Sanchis, A.J. Ramos * E. Pardo, S. Mar
Food Technology Department, Lleida University, Rovira Roure 191, 25198 Lleida, Spain Received 23 June 2003; received in revised form 28 January 2004; accepted 5 February 2004

Abstract Ochratoxin A (OTA) is a secondary metabolite of Aspergillus and Penicillium species, including Aspergillus ochraceus, a species that can be found in stored cereal grains such as barley. The objective of this study was to determine the effects of water activity (aw, 0.80 0.99), temperature (10, 20, 30 jC), and A. ochraceus isolate differences on radial growth and OTA production in irradiated barley grains. The three isolates showed optimal conditions for growth and ochratoxin A production at 0.99 aw and 30 jC, with a marked decrease of growth rates and OTA production at the lowest levels of aw and temperature assayed. The minimum aw level for growth, observed in this study, was 0.85 and 0.90 aw for OTA production. Significant differences among the isolates were found. Lag phases prior to fungal growth and OTA production values were modelled by multiple linear regression and response surface models. These models could provide an approximate prediction of growth and OTA production. D 2004 Elsevier B.V. All rights reserved.
Keywords: Aspergillus ochraceus; Barley grain; Ochratoxin A; Predictive modelling; Temperature; Water activity

1. Introduction Aspergillus ochraceus is important in stored grain because of its ability to produce ochratoxin A (OTA) (Van der Merwe et al., 1965), a potent nephrotoxin known also to be teratogenic, immunosuppressive and carcinogenic. It has been classified by the International Agency for Research on Cancer (IARC, 1993) as a possible human carcinogen (group 2B) based on sufficient evidence of carcinogenicity for animals
* Corresponding author. Tel.: +34-973-702811; fax: +34-973702596. E-mail address: ajramos@tecal.udl.es (A.J. Ramos). 0168-1605/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2004.02.003

and inadequate evidence in humans (Kuiper-Goodman, 1996). Barley grain is commonly colonized by toxigenic A. ochraceus together with various species of Penicillium, Fusarium, and yeasts (Clarke and Hill, 1981; Hill and Lacey, 1983; Sala, 1993). Some studies have shown the presence of OTA-producing isolates of A. ochraceus in stored barley grains contained by OTA (Cvetnin and Pepeljnjak, 1990). Individual fungal species differ in their growth responses to the water activity (aw) and temperature of the grain (Ayerst, 1969; Mislivec and Tuite, 1970; Magan and Lacey, 1988). Fusarium spp. are generally considered to be field fungi with a high water

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requirement for growth, although they can sometimes grow in stored grain, while Aspergillus and Penicillium spp. are typical storage species that are able to thrive at relatively low water activities (Ramakrishna et al., 1996). Exposure of grains to high moisture levels is most likely to occur during harvest, but may occur at other stages between harvest and final consumption. Mycotoxin contamination of grain is difficult to predict because it depends on a complex interaction of factors, such a temperature, moisture, kind of grain, endogenous fungal species, storage history, storage time, type of transit and transit time (Chelack et al., 1991). According to recommendations by FAO (JECFA, 2002) studies should be conducted to improve the understanding of the occurrence and ecology of the fungi that produce ochratoxin A. Growth of fungi has frequently been studied on nutrient media adjusted to different aw with glycerol and incubated at different temperatures. However, results obtained on culture media cannot necessarily be extrapolated to natural systems, and colonization patterns may be modified in grain ecosystems (Magan and Lacey, 1984, 1985). The moisture content and temperature are the most important variables in determining growth and rate of mycotoxin production by fungi (Magan and Lacey, 1988). The objectives of this study were (a) to determine the effects of water activity, temperature and time on growth and ochratoxin A production of three ochratoxigenic isolates of A. ochraceus on barley grains and (b) to obtain models for prediction of growth and OTA production as a function of aw and temperature which may be useful for predicting safe storage conditions for cereals.

2.2. Source of barley grain and gamma-irradiation treatment Spanish barley grain (variety Grafic) harvested in 2001 was used and exposed to 12 kGy gamma irradiation and stored at 4 jC. This dose was sufficient to kill all fungi, bacteria and yeasts on or within the grain without affecting seed germination (Ramakrishna et al., 1991).

2. Materials and methods 2.1. Fungal isolates Three ochratoxigenic isolates of A. ochraceus Wilhem ( = Aspergillus alutaceus Berk & Curt) isolated from cereals were used: isolate NRRL 3174 (coded as 3.94) and isolates 3.113 and 3.38, which are deposited in the Food Technology Department Collection of the University of Lleida, Spain.

Fig. 1. Effect of water activity and temperature on growth rates of three OTA-producing isolates of A. ochraceus on barley grains. Different letters over bars mean significant differences between fungal diameter at different conditions set (Least Square Means Test adjusted by Tukey Kramer, with significant level of 0.05). (N.G.: no growth).

E. Pardo et al. / International Journal of Food Microbiology 95 (2004) 7988 Table 1 Minimum water activity for growth/OTA production, at different temperatures, for the three isolates of A. ochraceus on barley grains after 28 days incubation Isolate Temperature 10 jC 3.113 3.38 3.94 / 0.95/ 0.95*/0.95* 20 jC 0.85/0.95 0.85/0.90* 0.85/0.90 30 jC 0.85/0.95 0.85/0.95 0.85/0.90

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previously modified with glycerol to the required aw. Thoma chamber was used to determine the final spore concentration, which was in the range 1 5 106 spores ml 1. 2.5. Inoculation and incubation Grains were aseptically transferred into 9 cm diameter sterile plastic Petri plates (22 g/plate) forming a continuous layer of grains and each plate was point-inoculated in the centre with 10 Al of spore suspension. Plates with the same conditions were enclosed in sealed containers along with beakers containing glycerol water solutions of the same aw as the plates in order to maintain an atmosphere with the same equilibrium relative humidity (Dallyn, 1978), and incubated at 10, 20 and 30 jC. All treatments were repeated twice and the experiment was carried out for 28 days. 2.6. Measurement of mycelial growth The Petri plates were examined daily or as required, and two diameters at right angles were measured of each colony. The increase in radial growth was determined and used to calculate the growth rate (mm day 1) by linear regression, under each set of treatment conditions for each isolate. 2.7. Determination of ochratoxin A production Samples were incubated for 4 weeks, and then frozen ( 20 jC) until OTA extraction and analyses

No growth/no OTA production. * Growth/OTA production only occurred under that condition.

2.3. Water activity adjustment of grain Water activity was adjusted to 0.80, 0.85, 0.90, 0.95 and 0.99 by aseptically adding amounts of sterile distilled water (0.63, 2.18, 5.31, 11.87, and 29.37 ml water/ 100 g of barley, respectively) to the gamma-irradiated grain in sterile 1-l flasks. The wetted grain was then equilibrated for 48 h at 4 jC with periodic shaking by hand during this time. The amount of water necessary to produce the required water activities was determined by a moisture absorption curve previously made for the same barley. Final aw values were checked with a water activity meter (AquaLab, Pullman, WA, USA). 2.4. Inoculum preparation Cultures of three isolates were incubated at 25 jC for 7 days on a barley-based medium (Ramos et al., 1998). Spores were suspended in sterile distilled water containing 0.005% Tween 80. Of this stock spore suspension, 1 ml was added to 2 ml of sterile water

Table 2 Effects of temperature and water activity on growth and production of ochratoxin A of three isolates of A. ochraceus on barley grains Source of variation Growth study DF Isolate (I) Temperature (t) aw I aw t aw It I t aw 2 2 3 6 6 4 12 MS 1559.6954 55,427.0140 26,368.3175 240.1747 6751.1866 343.6366 54.3901 F value 64.24** 2282.8** 1086.04** 9.89** 278.06** 14.15** 2.24* OTA production study DF 2 2 2 4 4 4 8 MS 1.1879 76,508 308,374 308,182 218,806 764,790 218,700 F value 315.45* 203.16* 81.89* 81.83* 58.10* 203.08* 58.07*

MS = mean square; DF = degrees of freedom. * Significant at p < 0.001. ** Significant at p < 0.0001.

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were carried out. The production of OTA in barley grain by three isolates of A. ochraceus was determined by high performance liquid chromatography (HPLC) according to methodology of Entwisle et al. (2000). The whole barley samples were ground and 10 g were extracted with 40 ml of acetonitrile (Merck, Darmstadt, Germany)/water solution (60 + 40), followed by shaking on a vortex for 2 3 min. The extract was filtered through Whatman no. 4 filter paper, 4 ml of the filtrate were taken and added to 44 ml of phosphate buffer saline (PBS) pH 7.4. The mix was cleaned-up by an immunoaffinity column ne diagnostics Technology, Glasgow, (Ochraprep, Rho Scotland) at a flow rate of 2 3 ml min 1. The columns were then washed with 20 ml of PBS at a flow rate of 5 ml min 1 and finally dried in an air stream. Desorption was carried out with 3 ml of acetic ac. (Prolabo, Briare Le Canal, France)/methanol (Merck)/water (1:49:50) solution, slowly passed through the column. Finally, 100 Al of OTA extract was quantified by HPLC. The HPLC system was equipped with a 474 fluorescence detector (Waters, Mildford, MA, USA), (kexc 333 nm; kem 443 nm) and a C18 column (Waters Spherisorb 5 Am, ODS2, 4.6 250 mm) all under control of Waters Millenium32 software. The analysis was performed under isocratic conditions at a flow rate of 1 ml min 1 of the mobile phase (acetonitrile/ water/acetic acid; 51:47:2). OTA was quantified by the external standard method. The ochratoxin standard was from A. ochraceus (Sigma-Aldrich, St. Louis, MO, USA). The standard solution was made in toluene (Merck)/acetic ac. according to the concentration established using an UV spectrophotometer. The required standard quantity was evaporated to dryness and dissolved in the mobile phase in order to prepare 10 solutions in the range of concentration of OTA from 0.0005 to 5 Ag l 1. The calibration curve obtained proved linear. The retention time of OTA under the conditions described was approximately 8.5 min, and the detection limit for OTA was 1.5 ng g 1 barley. 2.8. Statistical analysis of the data In the fungal growth experiment, the variable measured was the colony diameter at different aw/ temperature versus time. Linear regression of increase

in radius versus time was used to obtain growth rates under each set of conditions. The program Microsoft Excel version 97 was used for this purpose. Analysis of variance for the different sets of growth results and for the amount of OTA detected in samples were carried out using the SAS version 8.2 (SAS Institute, Cary, NC, USA). 2.9. Obtention of models by RSM Lag phases and OTA production values were described by a polynomial model equation (MLR) with

Fig. 2. Effect of water activity and temperature on OTA production by three isolates of A. ochraceus on barley grains. (N.P.: no production). Limit of detection = 1.5 ng g 1 barley.

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six coefficients (b0, b1, b2, b12, b11 and b22): lag phase/ OTA production = b0 + b1T + b2aw + b12Taw + b11T 2 + 2 b22aw , and the resulting response surface models (RSM) were obtained with the UnscramblerR version 7.6 (CAMO ASA, Oslo, Norway), including the significant factors, interactions and quadratic terms.

3. Results 3.1. Growth study Optimal conditions for growth on barley grain of the three isolates of A. ochraceus tested were 30 jC and 0.99 aw (Fig. 1). At 0.80 aw none of the isolates grew. Minimum aw for growth was found at 0.85 at levels of temperature of 30 and 20 jC, and at 0.95 aw at 10 jC (Table 1). Factors assayed, water activity, temperature and isolates, as well as their two- and three-way interactions significantly affected colony radius, during incubation for 28 days (Table 2). At 30 jC, the decrease at water activity level from 0.99 to 0.95 resulted in a sharp reduction of the growth rates (Fig. 1). At the remaining aw levels this decrease was less marked. At 20 jC, the influence of the water activity level on fungal growth rates was less marked, while at 10 jC growth rates were slightly

higher at 0.95 than at 0.99 aw, although there were no significant differences. The influence of the temperature on mycelial growth was more marked at 0.99 aw (Fig. 1). Under these conditions the three isolates showed maximum growth rates at 30 jC, being 3.34, 3.92 and 4.42 mm day 1 for isolates 3.113, 3.38 and 3.94, respectively, decreasing at 20 jC, while at 10 jC isolates 3.94 and 3.113 did not grow. The trend was similar at 0.85 0.90 aw, while at 0.95 aw all the strains grew faster at 20 jC than at 30 jC. Intra-specific differences were found. At 10 jC isolate 3.38 grew at 0.95 and 0.99 aw, isolate 3.94 grew only at 0.95 aw and isolate 3.113 did not grow at all. Growth rates of isolates 3.38 and 3.94 were in general significantly faster than those of isolate 3.113, regardless of aw and temperature level. 3.2. OTA production study All single factors, temperature, water activity and isolate, and their two and three-way interactions had significant effects on OTA-production after incubation for 28 days (Table 2). Optimal conditions for OTA production for the three isolates were 0.99 aw and 30 jC. OTA production at 0.95 aw and 30 jC decreased markedly, while

Table 3 Model coefficients obtained by MLR for effects of aw and temperature on lag phases prior fungal growth (mm day 1) and OTA production on barley grains Isolates Factors Intercept (b0) Temperature (b1) aw (b2) T aw (b12) (T)2 (b11) (aw)2 (b22) R2
a

3.113

3.38

3.94

3.113

3.38 39.785# 0.256# 36.553# 1.924# 1.354# 0.688# 0.327

3.94 6.84 106** 4.69 104** 6.49 106** 2.91 105** 2.21 105* 1.02 105# 0.923

Regression coefficients growth studya 106.498** 1.040** 86.804** 3.722** 5.455** 3.940** 0.915 113.935** 0.893** 99.266** 2.063* 4.227** 4.301** 0.883 107.822** 0.937** 92.197** 2.692* 4.477** 5.016** 0.879

Regressions coefficients OTA production studyb 1730* 6.670* 1727* 55.171* 1.092# 27.751# 0.437

Values of interaction and square effects: T aw = 0.1211(T 20) 14.554(aw 0.898); (T)2 = (0.1211(T 20))2; (aw)2 = (14.554 (aw 0.898))2. b Values of interaction and square effects: T aw = 0.1202(T 20) 26.653(aw 0.9467); (T)2 = (0.1202(T 20))2; (aw)2 = (26.653 (aw 0.9467))2. * Significant at p < 0.05. ** Significant at p < 0.0001. # Not significant.

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Fig. 3. Response surface contour plots showing the effect of aw and temperature on lag phase prior to fungal growth (days) of three ochratoxigenic isolates of A. ochraceus, on barley grains.

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Fig. 4. Response surface contour plots showing the effect of aw and temperature on OTA accumulation by three isolates of A. ochraceus on barley grains after 28 days.

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no production was found under the remaining aw levels. At 20 jC OTA was only produced at 0.95 0.99 aw by the isolates 3.94 and 3.113, while the isolate 3.38 only produced OTA at 0.90 aw. At 10 jC OTA was only produced by isolate 3.94 at 0.95 aw (Fig. 2). Significant intra-specific differences were found, due to the high production by A. ochraceus 3.94 when compared with the other two isolates. 3.3. Predictive modelling Table 3 shows the estimated model coefficients by the multiple regression analysis. Response surface contour plots showing the effects of water activity and temperature on the lag phases prior fungal growth and OTA production values are shown in Figs. 3 and 4. When no fungal growth was found, the lag phases values entered were the experiment duration: 28 days. Negative values in plots should be interpreted as null values.

4. Discussion This study has shown a significant influence of water activity and temperature on growth and ochratoxin A production on barley grains of three isolates of A. ochraceus. Minimum water activity level found to be 0.85 for growth of isolates of A. ochraceus and 0.90 aw for OTA production. Previous studies found a minimum water activity values of 0.77 for growth of isolates of A. ochraceus (Pitt and Christian, 1968; Kozakievicz and Smith, 1994) and of 0.80 0.87 aw for OTA production (Northolt et al., 1979; Adebajo et al., 1994; Kozakievicz and Smith, 1994). Optimum water activity level for OTA production by A. ochraceus isolates was found at 0.99 on malt and Czapek maize extract media (Northolt et al., 1979). These results were obtained on artificial substrates and may not accurately represent the real capacity of A. ochraceus to grow on a natural substrate. It has been suggested that nutrient source can affect the minimal aw for growth (Wearing and Burgess, 1979), as nutrients may be more readily available in artificial media. Studies of Christensen (1962) and Lopez and Christensen (1967) suggested that aw minima for growth of A.

ochraceus vary from 0.76 to 0.88 depending on substrate. The duration of the experiment could have an influence on minimum water activity level for fungal growth and OTA production. Most grains are stored considerably longer than 28 days, but under the experimental conditions, sterile grain with no other fungal or insect activity and without temperature fluctuations there is low probability for moulds to germinate if they have not germinated after 28 days. Optimal conditions for fungal growth on barley grain were 30 jC and 0.99 aw for the three isolates of A. ochraceus tested. This temperature level also supports the maximum growth of A. ochraceus at 0.96 0.98 aw suggested previously (Ramos et al., 1998). Interestingly, as temperature level decreased, the maximum growth was found at 0.95 0.99 aw. Optimum water activity level for OTA production on barley grain was found at 0.99. Similarly, other authors found optimum aw level for OTA production at 0.98 on barley grains (Ramos et al., 1998). A. ochraceus 3.94 was the only isolate which produced OTA at 10 jC, at a water activity of 0.95, although the concentration was low (5.14 Ag kg 1). Other authors showed no OTA production by isolates of A. ochraceus at 10 jC, in liquid cultures (Sansing et al., 1973) and on autoclaved barley grains (Ha ggblom, 1982; Damoglou et al., 1984). This suggests that temperature is a critical factor influencing ochratoxin production and has a pronounced influence in intra-specific differences production. Ha ggblom (1982) reported a substantial influence of temperature on OTA production by two isolates of A. ochraceus on autoclaved barley grains. This author suggested that production of OTA is not associated with rapid growth of the fungi, rather higher growth rates seem to restrict OTA production. Similarly, Madhyastha et al. (1993) found no direct relationship between fungal growth and ochratoxin production when A. ochraceus NRRL 3174 (3.94) was grown on autoclaved barley at 0.94 aw. Good correlation (R2 = 0.95) between growth rate and OTA production was obtained for A. ochraceus 3.113, obtaining increasing OTA yields with increasing growth rates. The other two isolates, 3.38 and 3.94, showed correlation coefficients of 0.62 and 0.54, respectively. This study shows that OTA production on barley occurs within a more restricted range of water activity and temperature than fungal growth. This point is

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important in order to maintain the appropriate conditions in stored barley that prevent toxigenic fungal growth and thus OTA production. With this aim the predictive models obtained can be extrapolated to stored barley. An empirical approach to modelling the effects of aw on mould growth was used by Gibson et al. (1994), who adjusted data to existing equations such as the square-root model (Ratkowsky et al., 1982) or Arrhenius equation (Schoolfield et al., 1981). Cuppers et al. (1997) used the Rosso and Ratkowsky models to describe the combined effects of temperature and NaCl on the growth rate of some food spoilage moulds. The need to quantify the effects of each of the factors contributing to the total microbiological integrity of products and the availability of powerful computers are increasing the use of mathematical modelling (Gibson and Hocking, 1997). In the study reported here, time to visible moulding can be obtained as a function of aw temperature, giving a guide to the time barley grain may be stored under certain conditions. Moreover, the model for OTA accumulation may be extended including the temporal variable and thus allow prediction of OTA accumulation as a function of time, aw and temperature. Another variable which is probably important when determining storability is initial inoculum (contamination), and this could also be included in such a model. Accompanying mycoflora may also play a role. All this can assist in carrying out hazard analysis and critical control point in the production, handling and storage of grain.

Acknowledgements The authors are grateful to the Catalonian govern General de Recerca) and to the ment (Direccio Spanish government (CICYT, AGL 2001-2974-C0502) for their financial support.

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