Fitting of colony diameter and ergosterol as indicators of food borne mould growth to known growth models in solid medium

Sonia Mar n, Dolors Cuevas, Antonio J. Ramos, Vicente Sanchis PII: DOI: Reference: To appear in: Received date: Revised date: Accepted date: S0168-1605(07)00464-3 doi: 10.1016/j.ijfoodmicro.2007.08.030 FOOD 4118 International Journal of Food Microbiology 30 June 2006 3 July 2007 10 August 2007

Please cite this article as: Mar n, Sonia, Cuevas, Dolors, Ramos, Antonio J., Sanchis, Vicente, Fitting of colony diameter and ergosterol as indicators of food borne mould growth to known growth models in solid medium, International Journal of Food Microbiology (2007), doi: 10.1016/j.ijfoodmicro.2007.08.030

This is a PDF le of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its nal form. Please note that during the production process errors may be discovered which could aect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
FITTING OF COLONY DIAMETER AND ERGOSTEROL AS INDICATORS OF FOOD BORNE MOULD GROWTH TO KNOWN GROWTH MODELS IN SOLID MEDIUM

Sonia Marn*, Dolors Cuevas, Antonio J. Ramos and Vicente Sanchis

Lleida, Spain

*Corresponding author. Mailing address: Food Technology Department, Lleida University, CeRTA-UTPV, Rovira Roure 191, 25198 Lleida, Spain. Phone: 34 973702555. Fax: 34 973702596. E-mail: smarin@tecal.udl.es

AC CE P

TE

MA

NU

SC

Food Technology Department, Lleida University, CeRTA-UTPV, Rovira Roure 191, 25198

RI

PT
1

Running title: Fitting mould growth to models

ACCEPTED MANUSCRIPT

ABSTRACT Growth of a range of 14 common food spoilage fungal species was evaluated along

Growth was assessed under different environmental conditions following a central composite design. The suitability of using either linear, Gompertzs or Baranyis models for primary

asymptotic Baranyis function gave better estimations of growth rate and lag phase when no asymptotic trend was observed. When a decrease in growth rate was observed with time, standard Baranyis model was chosen, although the search for new mechanistic models specific for moulds would probably improve the estimations. The use of Gompertz equation led, in general, to overestimated parameters. Ergosterol showed good performance as a

parameters may be useful for primary modelling and thus for subsequent secondary modelling.

Keywords: fungi, food spoilage, growth, modelling, sigmoidal

AC CE P

TE

coefficients were found between ergosterol and colony diameters, suggesting that both

fungal growth indicator for the whole range of species. Finally, significant correlation

MA

NU

SC

modelling of the results was tested. Regarding colony diameters, using either linear or

RI

PT

time as a function of both colony diameter and ergosterol content on malt extract agar.

ACCEPTED MANUSCRIPT
INTRODUCTION Predictive microbiology has traditionally dealt with prediction of food spoilage by bacteria. The non-pathogenic nature of moulds together with the lack of good methods for assessing

researches. However, moulds are important causative agents of economical losses mainly regarding cereals and their derivatives. In indoor environments fungal volatiles/mycotoxins

increasing concern regarding the presence of mycotoxins in foods, and the difficulty of eliminating them from the foodstuffs once produced, has highlighted the importance of preventing growth of toxigenic fungal growth as the main alternative for preventing mycotoxin contamination in foodstuffs.

Mould growth (expressed as increase in colony diameter) was first empirically

and filamentous fungi such as Penicillium roqueforti (Valik et al., 1999) and Aspergillus flavus (Gibson et al., 1994). The modified version of the Gompertz model (Zwietering et al., 1990) has been sometimes selected for empirically modelling mould growth on the basis of its proven flexibility to different asymmetrical growth data (Char et al., 2005). In addition, the maximum growth rate (m) estimated by a modelling step is equivalent to the slope of the straight line observed on the plot. From these primary models some parameters are usually estimated such as m and lag phase (), which are subsequently used for secondary modelling. Modelling of colony diameter may be useful for research purposes; however, it is not a measurable parameter in routine food analysis. Ergosterol analysis, which accounts for the total fungal population in food samples, may be an alternative, although its determination may be tedious and lengthy. In previous kinetic studies with Penicillium expansum, ergosterol determination showed lower repeatability and sensitivity than colony diameters measurements (Marn et al., 2006).

AC CE P

TE

bacterial growth. This model has been successfully used to fit growth of bacteria and yeast,

modelled against time using the model of Baranyi et al. (1993) originally developed for

MA

NU

SC

have been shown to provoke adverse health effects (Samson et al., 1994). In addition, the

RI

PT

their growth are the reasons why they have been neglected in predictive microbiology

ACCEPTED MANUSCRIPT
The objective of this study was to compare the commonly used growth models for primary modelling in order to assess their usefulness for estimation of growth parameters. Also the use of ergosterol, besides colony diameters, as mould growth indicator for primary

MATERIALS AND METHODS Fungal isolates

The strains belonged to 14 fungal species representing general common food contaminants found in intermediate moisture foods: Alternaria alternata (Fr.) Keissl. CECT2662, Aspergillus carbonarius (Bainier) Thom CECT2086, A. flavus Link CECT2687, A. ochraceus K. Wilh. NRRL3174, A. parasiticus Speare CECT2688, Cladosporium cladosporioides (Fresen.) G.A. de Vries CECT2110, Eurotium amstelodami L. Mangin CECT2586, Fusarium

Thom CECT2278, P. verrucosum Dierckx CECT2906, and Rhizopus oryzae Went & Prins. Geerl. CECT2339 (CECT: Spanish Type Culture Collection). Experimental design

Experimental runs were generated by a Central Composite Design (circumscribed, alpha=2) by means of The Unscrambler version 7.6. The factors entered were aw (0.85-0.95), temperature (15-30C), pH (5-7) and potassium sorbate concentration (0.5-1.5%), and the program generated a model with a centre point which was tested seven times as well as 24 test samples which were prepared and carried out in a random sequence (Table 1). The levels of the factors were chosen to simulate those of intermediate moisture foods kept at room temperature, thus susceptible of fungal spoilage. Five different sets of inoculated Petri plates were prepared which were assessed for fungal growth diameters periodically and analysed for ergosterol content when colonies attained approximately 15, 30, 45, 60 and 75

AC CE P

TE

racemosus Fresen. CECT2253, Penicillium chrysogenum Thom CECT2802, P. expansum

graminearum Schwabe CECT2150, F. verticillioides (Sacc.) Nirenberg 25N, Mucor

MA

NU

SC

RI

PT

modelling was assessed. Secondary modelling was not studied in the present work.

ACCEPTED MANUSCRIPT
mm of diameter (31 experiments x 14 isolates x 5 sampling times = 2170 ergosterol assays). Experiments lasted for no more than 6 months. Media Malt Extract Agar (MEA) was used as medium. The pH of the medium was adjusted to pH

while pH 8 was achieved by means of NaOH. Glycerol was added to the media in order to prepare media at 0.95, 0.90, 0.85 and 0.80 aw levels. Experimental curves were built in order

later purpose initial medium was prepared with different glycerol concentrations and final aw

glycerol added were plotted against aw and a suitable calibration curve was fitted. Finally, potassium sorbate was added at the required concentrations. All 31 media were prepared separately, autoclaved and plated onto 9-cm Petri plates. Water activity values were checked in the prepared plates, as well as pH (Crison micropH2000 pH meter, Crison, Barcelona,

Inoculation and incubation

The strains were grown on Malt Extract Agar (MEA) for 14 days and suspensions (105CFU ml-1) were prepared in 0.005% Tween 80 solutions. Petri plates were single point inoculated (101-102 CFU) in the middle for each fungal suspension. Petri plates with the same water activity level were enclosed in sealed polyethylene bags and placed in suitable incubators following the experimental design.

Measurement of colony diameters Periodically, daily or as required, Petri plates were observed and colony diameters recorded. When colonies achieved approximately 15, 30, 45, 60 and 75 mm of diameter, one Petri plate per experiment and strain (434 in total) was taken and analyzed for ergosterol content.

AC CE P

Spain).

TE

MA

measured in each case with an Aqualab (Decagon, Pullman, USA), subsequently g of

NU

to calculate the amount of buffer solutions and glycerol to be added to the media. For the

SC

RI

levels of 4-7 by using McIlvaines buffer consisting of 0.1M citric acid and 0.2 M Na2HPO4,

PT

ACCEPTED MANUSCRIPT

Ergosterol analysis Colonies (on agar) were cut in small squares and extraction carried out. A modification of the

found to be around 84% for the concentrations found in the study; limit of detection was of 12 g per plate. Briefly, fungal colony was extracted with 40 ml (or 20 ml in case of tiny

transferred to a screw cap tube and placed in a hot water bath (55-60 C) for 20 min. The tubes were then allowed to cool to room temperature. Three milliliters of water and 2 ml of hexane were added to the tubes, which were then agitated in a Vortex mixer for 1 min. After separation of layers, the upper layer (hexane) was transferred to a 10-ml vial. Hexane extraction was repeated twice using 2 ml each time. The extracts were combined and

Waters 515 isocratic pump (Waters Associated, Milford, MA), a Waters 717plus autoinjector, a Waters Spherisorb ODS2 C18 column (4.6 x 250 mm). The Waters 2487 variable wavelength UV detector was set at 282 nm. The mobile phase was methanol at 1 ml min-1. Ergosterol standard was purchased from Sigma (St. Louis, Mo) for calibration line (R2=0.99). Statistical analyses

From the 434 experiments, those carried out with Fusarium species, C. cladosporioides, A. alternata and A. carbonarius led to no-growth after 180 days in more than 74% of the cases, and erratic under most of the remaining conditions, thus the authors decided not to use them in this study. From the remaining strains (279 experiments), 142 experimental data corresponded to growth kinetics, 137 to no-growth after 180 days. Root-square ergosterol content and colony diameters were plotted against time. For each treatment, diameters were adjusted to the sigmoidal Baranyis function [1] (Baranyi et al., 1993), and to the Gompertz model modified by Zwietering et al. (1990) [2] by using

AC CE P

TE

methanol, and forced through 0.45 m acetate filters. The HPLC equipment consisted of a

evaporated to dryness under a stream of nitrogen. The dry extracts were dissolved in 2 ml of

MA

NU

SC

colonies) of 10% KOH in methanol by magnetically stirring for 30 min. A 10-ml aliquot was

RI

PT

method by Gourama and Bullerman (1995) was applied. Recovery rates were calculated and

ACCEPTED MANUSCRIPT
Statgraphics Plus 5.1; also the linear model was adjusted using Microsoft Excel 2000. Additionally, a biphasic Baranyis function was used in which the logarithmic term where Dmax appears was deleted in order to omit the upper asymptote, as suggested by Valik et al.

where A = t + t=time (d)

m= maximum growth rate (mm d-1) = lag phase before visible growth (d)

Dmax= maximum diameter attained, in most of the cases, the diameter of the Petri dish

[2]

[3] colony diameter = m * t +

Maximum growth rates, lag phases and final diameter attained were estimated from the sigmoidal models, while maximum growth rates and lag phases were estimated from the linear and Baranyis biphasic ones. In the linear case, lag phase was estimated through the intercept of the regression line with the X-axis.

RESULTS Primary modelling of colony diameters: comparison of sigmoidal, linear and Baranyis biphasic models Firstly, modelling was carried out in colony diameter data. In general, data plots showed, after a lag phase, a linear trend with time. Only treatments 4, 13, 19 and 31 (0.85 aw) and

AC CE P

TE

m * e colony diameter = Dmax * exp exp * ( t ) + 1 D max

1 * log[exp( m * t ) + exp( m * ) exp( m * t m * )] m

MA

NU

SC

1 * log[exp( m * t ) + exp( m * ) exp( m * t m * )] m

RI

[1]

exp( m * A) - 1 colony diameter = m * A - log 1 + exp(D max )

PT
7

(1999) [3].

ACCEPTED MANUSCRIPT
those at 0.90 aw and 22.5C led in some cases to asymptotic curves (Fig. 1, 2), due to the difficulty of maintaining a constant aw for such long periods of time. Thus under normal circumstances convergence of the sigmoidal models relied on the diameter of the Petri dish

growth functions in this case as the upper asymptote is an external parameter. Easier convergence was found when using modified Gompertz model, than with Baranyis one.

important due to the poor relevance of this parameter for practical issues, as long as it does not interfere in m and estimations. Regarding linear model, plotting of the results against time and manual (and subjective) selection of the straight part of the line (avoiding lag phase and asymptotic one, if any) was required. Finally, Baranyis biphasic model seemed to be the best choice in most of the cases (no subjectivity involved). R2 obtained for the 31

known that, depending on the distribution of the experimental points, good R2 do not necessarily mean accurate estimation of parameters. In general, higher growth rates and lag phases were obtained when using modified Gompertz model than with the others, the confidence intervals (P<0.05) being wider (Table 2). 10 sets of data were generated through either Baranyis biphasic, Baranyis sigmoidal and modified Gompertz functions following a normal distribution of the data around each mean with variance equal to 1. Estimation of m and and their confidence intervals through Gompertz, Baranyis and linear models (Fig. 3) revealed that modified Gompertz model overestimated both m and when data were generated by any of the others. The linear model gave reasonably accurate estimations of both parameters generated by the Baranyis biphasic one. In this case, Baranyis model did not converge to any estimated parameters due to the lack of asymptotic data, while it lead to underestimation of parameters when data were generated with Gompertzs one. Thus, in the absence of an upper asymptote, Baranyis

AC CE P

TE

for the Gompertz, Baranyi, biphasic Baranyi and linear models, respectively. However, it is

treatments and 9 strains ranged from 94 to 100%, 87 to 99%, 94 to 100%, and 78 to 100%

MA

NU

SC

However, the former led to overestimations of the final diameter, although this point is not

RI

PT

as upper asymptote; this means that the use of these models has no biological support as

ACCEPTED MANUSCRIPT
biphasic model is the best alternative, while complete Baranyis model may be the best when an asymptotic stage is achieved. Fig 4 shows two examples of the variability of the observed data and their respective

to biphasic Baranyis model. Coefficients of variation of the observed values along time were high around the lag phase but they decreased to less than 18% after 13-15 days of

Primary modelling of ergosterol content

Ergosterol content did not show an exponential trend versus time, but a potential one (Fig 5). Thus log transformation of the data was less useful than root-square one, which led to both homogenisation of variance and linearisation of the data versus time. Data were

time, and this resulted in high error levels of the estimations when compared to diameter models (Table 2 and 3). Fig 5 also shows two examples of the variability of the observed data for the 7 replicates of the centerpoint adjusted to either standard or biphasic Baranyis model.

Fig 6 shows the experimental points adjusted to either standard or biphasic Baranyis model for 4 different treatments. Ergosterol accumulation along time followed a similar trend to diameter increase for the different treatments.

Correlation between colony diameters and ergosterol content Significant Pearson correlation coefficients were found between colony diameters and rootsquare (ergosterol content) of colonies of each of the strains tested under the 31 treatments tested ranging from 0.66 to 0.91. The rate root-square(ergosterol)/diameter was rather constant (between 0.20-0.24 for Aspergillus species, 0.18-0.21 for Penicillium species, 0.16

AC CE P

TE

set of data. In this case regression analyses were carried out with 6 observed values over

subsequently adjusted to either Baranyis or Baranyis biphasic model, depending on each

MA

NU

SC

incubation.

RI

PT

adjusted functions for the 7 replicates of the centerpoint (0.90 aw, 22.5C, pH 6, 1%) adjusted

ACCEPTED MANUSCRIPT
for E. amstelodami, while R. oryzae and M. racemosus, with a few observations, showed rates of 0.13 and 0.29, respectively) (Fig. 7).

One challenge when working with moulds is their explorative and exploitative nature for colonising solid substrates (Robinson et al., 1993), thus using liquid media for modelling their

used method to assess mould growth in solid substrates is colony diameter measurement, with some authors using CFU counts (Vindelov and Arneborg, 2002), although it has been shown that this latter parameter is linked to sporulating abilities of the species tested and has poor correlation to biomass weight (Marn et al., 2005). Although easily assessed, colony diameter measurement is difficult to be applied to real food substrates. Alternatively,

total fungal biomass has been shown to be correlated to biomass dry weight for the same strains tested in the present study; a ratio between 0.6 to 1.6 mg dry biomass per g ergosterol was found depending on the species tested (Marn et al., 2005). In this study the total pool of data obtained represent most of the situations one can encounter when dealing with fungal growth kinetics (from optimum growth to no-growth, through erratic growth under certain limiting conditions). The responses of the 9 strains to the 31 treatments assayed were observed to be either sigmoidal or just have a lag phase followed by a linear one. In general mould colony diameters have been primary modelled in the past by Baranyis, linear and modified Gompertz models, both linear and Baranyi approaches being the most common, but no study has compared before the usefulness of each of them. This study shows that under favourable growth conditions, the linear model may be the best alternative, however if the experimental design includes suboptimal conditions, the lag phase makes the linear model not convenient. On the other hand, both Baranyi and Gompertz models have been taken from bacterial growth curves in which log

AC CE P

TE

(Gourama and Bullerman, 1995; Saxena et al., 2001). This parameter, which accounts for

ergosterol content has been used for mould contamination in cereals and other food products

MA

NU

SC

growth (as generally done for bacteria) is not realistic. In the literature, the most common

RI

PT

DISCUSSION

10

ACCEPTED MANUSCRIPT
(N/No) is plotted against time in the presence of limited nutrients; thus after an initial lag phase, an exponential multiplication of bacterial cells is observed after which, and due to the shortage of nutrients or the accumulation of toxic metabolites the growth rate becomes

plotting diameters (or radiuses) of a mould colony growing in an agar Petri plate against time, a lag phase is observed, followed by a linear phase, but in most of the cases no decrease in

conditions a fungal colony would then probably grow indefinitely, if the agar plate was unlimited; thus, in the absence of the edge of the plate, the Baranyis biphasic model would be probably the best one (or the linear one in the absence of lag phase). This hypothesis contradicts the results obtained by Valik et al. (1999), who described for Penicillium roqueforti diameter a growth curve typical of microbial growth with lag-phase, linear phase

Equivalent estimated parameters (m, ) were found using the different models except for Gompertz one which was shown to overestimate both of them. Sigmoidal models other than the ones traditionally used may have better potential to describe fungal growth process. Under many circumstances, primary modelling yields reliable information on the value of m, but poor estimates are often obtained for the lag time (McKellar, 1997). Ergosterol was primary modelled for a range of food borne moulds for the first time. Mould colonies, while growing at a constant growth rate in diameter, yield by branching an exponential amount of biomass and consequently, of ergosterol amount. Thus ergosterol content over time should show a lag phase, followed by an exponential increase phase, and if log transformed, the curve should show a lag phase followed by a linear increase. In our case, when ergosterol results were log transformed, a final phase in which ergosterol did not increase exponentially anymore or even stopped was observed, while a clear decrease in diameter growth rate was not observed, suggesting that even the colony extended in area, a decrease in the rate of ergosterol accumulation occurred. This supports the work by Koch

AC CE P

TE

12 cm, well before the edge was reached.

and upper asymptote when using 17 cm Petri plates with asymptotic values between 2 and

MA

NU

SC

growth rate is observed before the edge of the Petri plate is reached. Under constant

RI

PT

constant and eventually decreases. In this study, and from our previous experience, when

11

ACCEPTED MANUSCRIPT
(1975), who reported that moulds form mycelium whose weight, except at the early stage of growth, does not increase exponentially. For this reason, the square-root transformation was chosen in the present work for data transformation in order to homogenise variance of

A previous work on growing colonies of P. expansum and relationship with ergosterol accumulation showed that ergosterol determination leaded to increased differences among

variation within replicates observed for colony diameters and ergosterol content was similar, suggesting that the latter could be a good alternative. Also primary modelling of both squareroot ergosterol and colony diameters led to similar R2 values. Primary models are the first step for estimation of parameters such as m, or time for a certain colony size to be observed. Time for visible growth can also be directly observed

regression process. Dantigny et al. (2002) stated that lag time coincided with the completion of the germination process; say more than 99% of germination. Germination has also been studied by mycologists, from the predictive point of view, because prevention of germination invariably leads to prevention of growth. However studies are lengthy and tedious, thus prediction of lag time for growth would substitute parameters such as lag phase for germination, germination rate and time for 10 or 90% germination. This work has shown that, among the commonly used models, all of them showing similar goodness of fit, Baranyis model, either biphasic or not, depending on the observed kinetics, may be the best alternative for an accurate estimation of m and . Both growth rate and lag phase have been used in the past for secondary modelling; m being used for secondary modelling in about 75% of the publications. m may be a useful parameters to compare treatments, and also complements lag phase data. However, when dealing with prediction and prevention of fungal growth in food products, two parameters could be considered: i) the lapse of time to reach a visible colony which makes a product rejectable,

AC CE P

TE

values, the latter being slightly higher, and the former being always estimated through a

with no need for primary modeling. Lag phase and time for visible growth should have similar

MA

NU

SC

replicates compared to diameter measurements (Marn et al., 2006). However, in this study,

RI

PT

ergosterol data.

12

ACCEPTED MANUSCRIPT
and ii) the lapse of time to reach a colony size which might be conducive to mycotoxin accumulation; as it is not possible to establish such a correlation, in case of mycotoxigenic species any growth should be prevented. Thus in this context growth rate may be of poor

may be of interest. Secondary modelling will be the next step in this research. Also it is of great importance the validation of secondary models obtained in real food matrices.

ACKNOWLEDGEMENTS

This work was supported by the Spanish Government (CICYT, Comisin Interministerial de Ciencia y Tecnologa, project AGL 2004-06413/ALI, and Ramon y Cajal program).

REFERENCES

Char, C., Guerrero, S., Gonzalez, L., Alsamora, S.M., 2005. Growth response of Eurotium chevalieri, Aspergillus fumigatus and Penicillium brevicompactum in Argentine milk jam. Food Science and Technology International 11, 297-305. Dantigny, P., Soares Mansur, C., Sautour, M., Tchobanov, I., Bensoussan, M., 2002. Relationship between spore germination kinetics and lag time during growth of Mucor racemosus. Letters in Applied Microbiology 35, 395-398. Gibson, A.M., Baranyi, J., Pitt, I.J., Eyles, M.J., Roberts, T.A., 1994. Predicting fungal growth: the effect of water activity on Aspergillus flavus and related species. International Journal of Food Microbiology 23, 419-431. Gourama, H., Bullerman, L.B., 1995. Relationship between aflatoxin production and mold growth as measured by ergosterol and plate count. Lebensmittel-wissenschaft undtechnologie-foo 28, 185-189. Koch, A.L., 1975. The kinetics of mycelial growth. Journal of General Microbiology. 89, 209216.

AC CE P

TE

model bacterial growth. Food Microbiology 10, 43-59.

Baranyi, J., Roberts, T. A., McClure, P., 1993. A non-autonomous differential equation to

MA

NU

SC

RI

PT

application, and other parameters such as lag phase, time for a 2-3 mm visible colony, etc,

13

ACCEPTED MANUSCRIPT
Marn, S., Morales, H., Ramos, A.J. Sanchis, V., 2006. Evaluation of growth quantification methods for modelling of Penicillium expansum growth in an apple-based medium. Journal of the Science of Food and Agriculture (in press)

growth of food spoilage moulds in solid substrates. International Journal of Food Microbiology 99, 329-341.

kinetics. International Journal of Food Microbiology 36, 179-186. Nout, M.J.R., Bonants-van Laarhoven, T.M.G., de Jongh, P., de Koster, P.G., 1987. Ergosterol content of Rhizopus oligosporus NRRL 5905 grown in liquid and solid substrates. Applied Microbiology and Biotechnology 26, 456-461. Robinson, C.H., Dighton, J., Frankland, J.C., 1993. Resource capture by interacting fungal

1994. Health implications of fungi in indoor environments. Elsevier, Amsterdam, The Netherlands.

Saxena, J., Munimbazi, C., Bullerman, L.B., 2001. Relationship of mould count ergosterol and ochratoxin A production. International Journal of Food Microbiology 71, 29-34. Valik, L., Baranyi, J., Grner, F., 1999. Predicting fungal growth: the effect of water activity on Penicillium roqueforti. International Journal of Food Microbiology 47,141-146. Vindelov, J., Arneborg, N., 2002. Effects of temperature, water activity, and syrup film composition in the growth of Wallemia sebi: Development and assessment of a model predicting growth lags in syrup agar and crystalline sugar. Applied and Environmental Microbiology 68, 1652-1657. Zwietering, M.H., Jongerburger, I., Rombouts, F.M., Vant Riet, K., 1990. Modelling of bacterial growth as a function of temperature. Applied and Environmental Microbiology 56, 1875-1881.

AC CE P

TE

Samson, R.A., Flannigan, B., Flannigan, M.E., Verhoef, A.P., Adan, O.C.G., Hoekstra, E.S.,

colonizers of straw. Mycological Research 97, 547-558.

MA

NU

SC

McKellar, R.C., 1997. A heterogeneous population model for the analysis of bacterial growth

RI

PT

Marn, S., Ramos, A.J., Sanchis, V., 2005. Comparison of methods for the assessment of

14

ACCEPTED MANUSCRIPT
FIGURE CAPTIONS

Figure 1. Run1 (0.90 aw, 22.5C, pH 6, 1% sorbate) and run14 (1.00 aw, 22.5C, pH 6, 1%

models for A. flavus (), A. ochraceus ({), P. expansum (), P. verrucosum (U), E. amstelodami () and R. oryzae ().

Figure 2. Run3 (0.95 aw, 15C, pH 7, 0.5% sorbate) and run16 (0.95 aw, 30C, pH 7, 0.5% sorbate) colony diameters raw data and adjusted (a) Baranyi, (b), Gompertz, and (c) linear models for A. flavus (), A. ochraceus ({), P. expansum (), P. verrucosum (U), E. amstelodami () and R. oryzae ().

Baranyis function and (c) modified Gompertz model (m=3; =10) with data points distributed as a normal distribution with 1 variance. Error bars are the confidence intervals of the estimated parameters (P<0.05).

Figure 4. Colony diameters adjusted to Baranyis biphasic function for (a) A. parasiticus and (b) P. chrysogenum for the 7 replicates in the centerpoint (0.90 aw, 22.5C, pH 6, 1% sorbate).

Figure 5. Ergosterol raw data and effects of log and square-root transformations on data variance for (a) A. parasiticus and (b) P. chrysogenum for the 7 replicates in the centerpoint (0.90 aw, 22.5C, pH 6, 1% sorbate).

Figure 6. Run1 (0.90 aw, 22.5C, pH 6, 1% sorbate) , run3 (0.95 aw, 15C, pH 7, 0.5% sorbate) , run14 (1.00 aw, 22.5C, pH 6, 1% sorbate) and run16 (0.95 aw, 30C, pH 7, 0.5%

AC CE P

TE

models for 10 sets of raw data generated by (a) Baranyis biphasic function, (b) standard

Figure 3. Estimated m and through modified Gompertz (), Baranyis (z) and linear ()

MA

NU

SC

RI

PT

sorbate) colony diameters raw data and adjusted to (a) Baranyi, (b), Gompertz, and (c) linear

15

ACCEPTED MANUSCRIPT
sorbate) square-root ergosterol raw data and adjusted to Baranyis model for A. flavus (), A. ochraceus ({), P. expansum (), P. verrucosum (U), E. amstelodami () and R. oryzae ().

Figure 7. Relationship between colony diameter and square-root ergosterol content for (a) A. parasiticus and (b) P. chrysogenum.

AC CE P

TE

MA

NU

SC

RI

PT

16

ACCEPTED MANUSCRIPT
Table 1. Central composite design with 4 designed variables and 31 run samples. Run 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 Water activity 0.90 0.95 0.95 0.85 0.80 0.95 0.90 0.90 0.90 0.85 0.95 0.90 0.85 1.00 0.85 0.95 0.90 0.85 0.85 0.90 0.90 0.90 0.85 0.90 0.95 0.95 0.95 0.90 0.90 0.90 0.85 Temperature (C) 22.5 15.0 15.0 30.0 22.5 15.0 7.5 22.5 22.5 15.0 30.0 22.5 15.0 6 5 7 7 6 7 6 6 6 pH Potassium sorbate concentration (%) 1.0

PT RI

1.5 0.5 1.5 1.0 1.5 1.0 1.0 1.0 0.5 0.5 1.0 0.5 1.0 0.5 0.5 2.0 1.5 1.5 1.0 1.0 0.0 1.5 1.0 1.5 0.5 1.5 1.0 1.0 1.0 0.5

MA

D TE

22.5 30.0 30.0 22.5 15.0 15.0 22.5 22.5 22.5 30.0 22.5 30.0 15.5 30.0 22.5 37.5 22.5 30.0

AC CE P

NU

SC
5 5 6 7 6 5 7 6 5 7 6 6 6 5 6 5 5 7 8 6 4 7

17

ACCEPTED MANUSCRIPT

Linear run 1 14 3 A. och 16 13 31 1 14 3 P. ver 16 13 31 1 14 E. 3 S.E. 3.340.08 5.130.10 3.420.09 8.210.08 0.260.01 0.310.05 0.570.02 1.090.07 2.500.06 1.340.07 0.230.02 1.370.09 1.810.13 2.80 0.12 3.77 1.19 14.95 18.62 18.04 0.09 2.59 -3.15 2.41 23.63 5.03 R2 99.7 99.7 99.2 100.0 98.3 89.9 98.3 94.5 99.4 94.6 94.0 95.5 94.8

Baranyis biphasic S.E. 3.340.08 5.180.13 3.540.09 8.210.07 NC NC 0.570.02 1.090.08 2.860.17 1.320.07 NC NC 1.820.10 S.E. 2.800.32 0.210.19 4.220.30 1.190.05 NC NC R2 99.8 99.7 99.5 100 NC S.E. NC NC NC

Baranyi

RI

colony diameter by linear, Baranyis and Gompertz models for A. ochraceus, P. verrucosum, E. amstelodami and P. expansum.
Gompertz R2 NC NC NC NC 99.7 94.8 NC NC NC NC 98.4 99.1 NC S.E. 4.300.39 5.960.31 4.170.09 9.060.36 0.380.02 0.820.29 0.630.02 1.550.61 4.560.96 1.750.16 0.360.03 1.970.13 2.730.16 S.E. 4.650.57 0.940.32 5.890.19 1.620.16 26.382.21 26.192.12 23.302.10 9.7910.31 9.922.99 0.551.30 21.404.28 29.421.02 9.480.58 A S.E. 93.77.7 84.02.5 97.04.4 107.010.7 34.30.9 9.20.4 76.912.3 137.029.8 237.9102.3 65.24.7 44.73.4 88.94.4 88.93.7 R2 99.5 99.5 99.8 99.7 99.4 95.4 98.6 95.6 98.8 96.1 98.1 98.8 98.9

SC
NC NC NC NC NC NC NC NC NC

S.E.

NU

MA

NC

PT ED

0.330.01

21.411.47 24.822.47

NC

0.590.17 NC NC NC NC 0.290.02 1.700.09 NC

17.951.63

97.9

CE

0.131.57

94.5

3.560.69

97.0 94.8 NC NC 96.4

AC

-3.691.47 NC NC 5.071.05

11.253.95

26.861.04

PT
-

Table 2. Examples of the estimated parameters (, growth rate, mm d-1; , lag phase, d; A, upper asymptote value, mm) through modelling of

A S.E. NC NC NC NC 83.30.4 9.30.4 NC NC NC NC 42.12.0

83.31.7

NC

18

ACCEPTED MANUSCRIPT

13 31 1 14 P. exp 3 16 13 31

1.260.09 1.180.04 4.700.62 2.690.09 1.080.01 -

7.39 8.17 0.76 4.48 -3.69 -

98.2 99.1 90.4 99.2 99.6 --

NC NC 3.330.38 3.160.29 1.190.05 -

NC NC -1.681.47 5.451.02 -2.031.11 -

NC NC 92.4 94.3 96.5

1.530.08 1.200.03 NC NC

PT

ams

16

6.210.19

2.11

99.3

6.0410.18

1.850.23

99.3

NC

NC

NC -

NC 99.5 99.6 NC NC NC -

6.630.29 1.930.27 1.480.05 5.120.76 10.727.78 1.20.045 -

2.530.32 16.832.17 13.470.73 1.290.97 22.7211.65 -1.280.76 -

111.619.3 84.87.9 76.91.7 86.38.6 884.2922.1 67.56.4 -

99.2 98.6 99.7 95.8 98.8 98.6 -

13.791.40

SC
NC NC NC -

8.730.74

RI

83.31.7

74.01.4 NC NC NC -

S.E., standard error of the estimated parameters -, no growth NC, no convergence

AC

CE

PT ED

MA
-

NC

NU

19

ACCEPTED MANUSCRIPT
Table 3. Estimated parameters (, increase in root-square ergosterol per day; , lag phase till ergosterol detection and A, maximum asymptotic value of root square ergosterol) through modelling of root-square ergosterol by Baranyis (either biphasic or

PT
NC -

standard) model for A. ochraceus, P. verrucosum, E. amstelodami and P. expansum.

1 14 3 A. ochraceus 16 13 31 1 14 3 P. verrucosum 16 13 31 1 3 14 16 31 1 3 16 13 31 -, no growth NC, no convergence 14 P. expansum

1.470.07 1.300.28 0.560.04 1.160.07 2.690.34 0.240.08 0.440.10 0.270.05

5.280.28 -0.151.47 4.830.94 0.810.35 8.890.39 -

RI

run

S.E.

S.E.

AS.E.

R2 99.9 97.8 98.6 99.3 99.7 84.2 95.5 100.0 99.2 NC 93.6 91.5 98.5 100.0 89.9 98.2 98.1 98.9 -

17.950.22

NU
NC -

MA

23.1614.28 1.044.33 12.861.51 12.670.53 -2.440.25

0.270.00

-6.723.90

TE

NC -

E.

AC CE P

0.420.13 0.210.04 -

22.747.65 -1.526.72 0.250.75 15.090.80 14.6912.49 2.090.79 3.381.62 -10.544.00

amstelodami

0.780.07

13

0.570.04 0.740.67 0.510.05 0.340.04 -

2.040.31

S.E., standard error of the estimated parameters

SC

20.951.41

20.590.50

18.732.42

15.170.12 16.841.81 25.361.67 19.121.58

20

ACCEPTED MANUSCRIPT

AC CE P

TE

MA

NU

SC

RI
21

PT

ACCEPTED MANUSCRIPT

AC CE P

TE

MA

NU

SC

RI
22

PT

ACCEPTED MANUSCRIPT

AC CE P

TE

MA

NU

SC

RI
23

PT

ACCEPTED MANUSCRIPT

AC CE P

TE

MA

NU

SC

RI
24

PT

ACCEPTED MANUSCRIPT

AC CE P

TE

MA

NU

SC

RI
25

PT

ACCEPTED MANUSCRIPT

AC CE P

TE

MA

NU

SC

RI
26

PT

ACCEPTED MANUSCRIPT

AC CE P

TE

MA

NU

SC

RI
27

PT