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Water activity and temperature effects on growth of Aspergillus niger, A. awamori and A. carbonarius isolated from different substrates in Argentina
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International Journal of Food Microbiology 119 (2007) 314 – 318 www.elsevier.com/locate/ijfoodmicro
Water activity and temperature effects on growth of Aspergillus niger, A. awamori and A. carbonarius isolated from different substrates in Argentina Andrea Astoreca a,⁎, Carina Magnoli a,c , María L. Ramirez a,c , Mariana Combina b,c , Ana Dalcero a,c a
Departamento de Microbiología e Inmunología, Facultad de Ciencias Exactas, Físico, Químicas y Naturales, Universidad Nacional de Río Cuarto, Ruta Nacional No. 36 Km 601, (5800) Río Cuarto, Córdoba, Argentina b Instituto Nacional de Tecnología Agropecuaria (INTA), Luján de Cuyo, Mendoza, Argentina c Member of the Research Career of CONICET, Argentina Received 28 May 2007; accepted 28 August 2007
Abstract The objectives of this study were to determine the effect of water activity, temperature, and their interactions on a) mycelial growth rate and b) the lag phase prior to grow of seven isolates of Aspergillus section Nigri isolated from peanuts, maize kernels, dried grapes and coffee cherries from Argentina. Three Aspergillus niger, three A. awamori and one A. carbonarius isolates examined showed optimum aW level for growth at 0.97 with optimal temperature of 30 °C. for most of the isolates and 25 °C for only one (A. awamori RCP176). Minimal aW for growth was 0.85 at the highest temperature tested. Overall growth was reduced up to 50% at 0.93 aW. Growth was also to a large extend inhibited at 0.85 aW for most isolates even after 21 days of incubation at temperatures lower than 30 °C. The analysis of variance of the effect of single (isolate, aW and temperature), two- and three-way interaction showed that all factors alone and all interactions were statistically significant (P b 0.001) in relation to growth rates and lag phase for A. niger, A. awamori and A. carbonarius isolates. These data are relevant since these species are isolated in high frequency on numerous substrates for human and animal consumption in Argentina. © 2007 Elsevier B.V. All rights reserved. Keywords: Aspergillus section Nigri; Fungal growth; Water activity; Temperature
1. Introduction Ochratoxin A (OTA) is receiving increasing attention worldwide because of the hazard it poses to human and animal health. The toxicological profile includes teratogenesis, nephrotoxicity and immunotoxicity. This toxin has been classified as a possible human renal carcinogen (group 2B) (International Agency for Research on Cancer, 1993). OTA is a toxin naturally produced by Aspergillus ochraceus and some species of Aspergillus section Nigri formerly A. niger group and A. carbonarius. These ochratoxigenic species have been reported in and upon a great variety of substrates from regions with warmer and tropical climate. They are able to tolerate diverse ⁎ Corresponding author. Tel.: +54 358 4676429; fax: +54 358 4676231. E-mail address: [email protected] (A. Astoreca). 0168-1605/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2007.08.027
conditions of moisture, pH and temperature (Pitt and Hocking, 1997; Abarca et al., 2001; Magan and Olsen, 2004). Ochratoxin A was until recently believed to be produced only by A. ochraceus and related species belonging to section Circumdati and by Penicillium verrucossum. However, some other Aspergillus has been reported as ochratoxigenic, numerous reports dealing with the production of OTA by members of Aspergillus section Nigri exist. In this section the reported OTA producing species are A. carbonarius and those now included in the so-called Aspergillus niger aggregate (i.e. A. awamori and A. niger) (Abarca et al., 2004). In Argentina, species included on Aspergillus section Nigri have been isolated in high frequency on wines grapes (Magnoli et al., 2003), dried vine fruits (Magnoli et al., 2004), corn kernels (Magnoli et al., 2006), peanut seeds and coffee cherries (personal communication). The ability of OTA production by
A. Astoreca et al. / International Journal of Food Microbiology 119 (2007) 314–318
these strains has also been demonstrated (Dalcero et al., 2002; Magnoli et al., 2003, 2004, 2006). Fungal growth results from the complex interaction of several factors (water availability, temperature and incubation time) and, therefore, an understanding of each factor involved is essential to understand the overall process and to predict fungal spoilage in agricultural and food products (Pardo et al., 2005). Little attention has been paid to the influence of incubation, temperature and changes of water activity either in media or in grains on fungal growth of A. niger and A. awamori. Marín et al. (1997) compared the effect of aW, temperature and their interaction on the germination rate, the lag phase and the mycelial growth profile of one isolate of A. niger on maize extract medium. Bellí et al. (2004) studied the effect of aW and temperature on mycelia growth of eight isolates of A. niger aggregate on a synthetic medium similar to grape composition. But, in the last study the authors did not discriminate which species among A. niger aggregate were used. The objectives of this study were to determine the effect of aW, temperature, and their interactions on a) mycelial growth
315
rate and b) lag phase prior to growth of seven isolates of Aspergillus section Nigri isolated from peanuts, maize kernels, dried grapes and coffee cherries from Argentina. 2. Materials and methods 2.1. Fungal species A total of seven isolates of Aspergillus section Nigri was used in this study. Two strains of A. niger (RCP42 and RCP176) isolated from peanut grains, two strains isolated from coffee beans: A. niger and A. awamori (RCC4 and RCC20, respectively), two strains of A. awamori isolated from maize kernels (RCM15 and RCM30) and one strain of A. carbonarius (RCDG30) isolated from dried grapes. These isolates were found to be OTA producers on YES medium (2% yeast extract, 15% sucrose) (Dalcero et al., 2002; Magnoli et al., 2003, 2004, 2006). The references in brackets are the codes of cultures held in the National University of Río Cuarto Collection.
▪
Fig. 1. Interaction of aW and temperature on the growth rate and lag phase of three isolates of A. niger, two on peanut extract medium (RCP42 and RCP176) and one on coffee extract medium (RCC4). Water activity levels: 0.85 (♦) 0.892 (□)—0.91(▴)—0.937(X)—0.955(⋄)—0.971( ) and 0.995 (▵).
316
A. Astoreca et al. / International Journal of Food Microbiology 119 (2007) 314–318
2.2. Medium The basic medium used in this study was a 3% (w/v) of each substrate (peanut, maize kernels, dried grapes and coffee beans) extract agar with a pH of 5.5. This was made by boiling 30 g of each ground substrate per litre in water for 45 min and filtering the resultant mixture through a double layer of muslin. The volume was made up to 1 l. The water activity of each basal medium was modified by the addition of known amounts of glycerol (Dallyn and Fox, 1980) to 0.85, 0.89, 0.91, 0.94, 0.95, 0.97 and 0.995. The water activity of representative samples of each medium was checked with an AquaLab Series 3 (Decagon Devices, Inc., WA, USA). Additional, control plates were prepared and measured at the end of the experiment in order to detect any significant deviation of the aW. 2.3. Inoculation and incubation Petri plates were inoculated centrally with a 4 mm diameter agar plug taken from the margin of a 7-day-old growing colony of each isolate on malt extract agar (MEA). Inoculated plates of the same aW were sealed in polyethylene bags. Triplicate sets of
each treatment were incubated at 15, 25 and 30 °C for 21 days. The experiment was repeated twice. Each isolate was only inoculated on the media, based on the substrate where it had originally been isolated. 2.4. Growth assessment Two perpendicular diameters of the growing colonies were measured daily until the colony reached the edge of the plate. The radii of the colonies were plotted against time, and a linear regression applied to obtain the growth rate (mm day− 1) as the slope of the line. Lag phase for growth was defined as the time (days) to reach 5 mm of diameter. 2.5. Statistical analysis In all cases, the linear regression of increase in radius against time (in days) was used to obtain the growth rates (mm day− 1) under each set of treatment conditions. The growth rates of each species were then evaluated by analysis of variance (ANOVA) using SigmaStat for Windows Version 2.03 (SPSS Inc.). When the analysis was statistically significant, the Tukey's multiple-
▪
Fig. 2. Effect of the interaction of aW and temperature on the growth rate and lag phase of three isolates of A. awamori, two on maize extract medium (RCM30 and RCM15) and one on coffee extract medium (RCC20). Water activity levels: 0.85 (♦) 0.892(□)—0.91(▴)—0.937(X)—0.955(⋄)—0.971( ) and 0.995 (▵).
A. Astoreca et al. / International Journal of Food Microbiology 119 (2007) 314–318
317
Fig. 3. Interaction of aW and temperature on the growth rate and lag phase of one isolate of A. carbonarius (RCDG30) on dried grapes extract medium.
comparison procedure test was used for separation of the means. Statistical significance was judged at the level P b 0.001. 3. Results Fig. 1 gives a diagrammatic representation of the interaction of aW and temperature on the growth rate (mm day− 1) and lag phase (h) of three isolates of A. niger, two on peanut extract medium (RCP42 and RCP176) and one on coffee extract medium (RCC4). The results showed that the maximum growth rates for the three isolates of A. niger were obtained at 0.97 aW and 30 °C. Temperature influenced the effect of aW on growth. The minimum aW for growth of the isolates of A. niger changed with temperature. At 0.85 aW – the lowest level of water stress tested – all the isolates were able to grow at 30 °C, although under this water availability the growth was very slow. When the temperature was 25 °C only one isolate (RCP42) was able to grow. At 15 °C, the growth rates of the isolates RCP42 and RCP176 were negligible when aW levels were low (0.85, 0.89, 0.91), increasing slightly at 0.94, 0.95, 0.97 and 0.995. A. niger RCC4 was unable to grow at any of the aW levels tested at 15 °C during the incubation period. Very short lag phases (b 24 h) occurred at 25–30 °C and 0.995 to 0.94. At lower temperatures the lag times increased
Table 1 Analysis of variance of effect of water (aW), temperature (T ), and different isolates (i), and their interactions on growth rate and lag phase of Aspergillus niger, A. awamori and A. carbonarius Source of variation
df
i aW T I × aW I×T aW × T I × aW × T
6 6 2 36 12 12 72
a
a
Growth rate MS
b
12.88 128.96 344.83 2.66 3.82 15.34 1.47
Degrees of freedom. b Mean square. c F-Snedecor. ⁎ Significant P b 0.001.
Lag phase F
c
111.60 ⁎ 1117.77 ⁎ 2988.90 ⁎ 23.06 ⁎ 33.15 ⁎ 132.94 ⁎ 12.74 ⁎
MS b
F
c
39339.53 839944.86 2972712.27 6836.45 31159.19 156942.20 7527.28
99.44 ⁎ 2123.22 ⁎ 7514.45 ⁎ 17.28 ⁎ 78.76 ⁎ 396.72 ⁎ 19.03 ⁎
considerably, especially at the low water activities assayed. Similar trends were found for all studied isolates. Fig. 2 shows the interaction effect of aW and temperature on the growth rate (mm day− 1) and lag phase (h) of three isolates of A. awamori, two on maize extract medium (RCM30 and RCM15) and one on coffee extract medium (RCC20). Aspergillus awamori strains RCM15 and RCM30 have an optimum aW to grow at 0.97 at 30 °C, whereas the optimum for the isolate RCC20 was 0.97 at 25 °C. The growth rates decreased as temperature changed from optimum to low marginal conditions. For the two isolates RCM30 and RCC20 and temperatures assayed, growth rates increased with aW, reaching the optimum at 0.97 aW and decreasing slightly at 0.995. The isolate RCM15 showed a similar behaviour at 30 °C but at 25 and 15 °C growth rates increased with aW, reaching the optimum at 0.995. At 15 °C, the growth of all the isolates was negligible when aWlevels were low (0.85, 0.89 and 0.91), increasing slightly at 0.94 to 0.995 aW. All the isolates were more tolerant to low aW at temperatures close to the optimum. The shortest lag phases for A. awamori were obtained at 0.995 to 0.94 aW at 30 °C. At marginal temperatures, the lag phases were markedly increased. Fig. 3 shows a diagrammatic representation of the interaction of aW and temperature on the growth rate (mm day− 1) and lag phase (h) of one isolate of A. carbonarius on dried grapes extract medium. The optimum aW for growth was 0.97 at all temperatures levels assayed, with the widest aW tolerance at 25–30 °C. The lag phase increased from b 24 h at 25–30 °C and 0.995–0.97 aW to N504 h at marginal temperatures and water availabilities. The analysis of variance of the effect of single (isolate, aW and temperature) two- and three-way interaction showed that all factors alone and all interactions were statistically significant (P b 0.001) in relation to growth rates and lag phase for A. niger , for A. awamori and A. carbonarius isolates (Table 1). 4. Discussion In the present study mycelial growth and lag phase prior to grow of seven isolates of Aspergillus section Nigri isolated from different substrates (peanut seeds, corn kernels, dried grapes and coffee beans) were found to be significantly
318
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influenced by aW, temperature and their interactions in vitro on agar based media. While the pattern of environmental factors effect was similar, the growth rates of the isolates varied significantly. Three A. niger, three A. awamori and one A. carbonarius isolates that were examined usually showed optimum aW level for growth at 0.97 with optimal temperature of 30 °C for most of the isolated and 25 °C for only one (A. awamori RCP176). Minimal aW for growth was 0.85 at the highest temperature tested. Overall growth was reduced up to 50% at 0.93 aW. Growth was also to a large extend inhibited at 0.85 aW for most isolates even after 21 days of incubation at temperatures lower than 30 °C. Lag times at the optimal temperature for A. niger, A. awamori and A. carbonarius isolates varied from 2 h at 0.97 aW to 260 h at 0.85 aW from 40 h at 0.97 aWto 235 h at 0.85 aW and from 2 h at 0.97 aW to 300 h at 0.85 aW, respectively, and increased by changing to marginal temperatures. Interspecies and intraspecies differences were found between the isolates, aW, temperature and their interactions. Few studies have examined the effect of different environmental conditions on the growth of Aspergillus section Nigri isolated from different substrates and most of these studies have been done on A. niger and A. carbonarius isolates. Bellí et al. (2004) studied the effects of water activity and temperature on growth of Aspergillus section Nigri (A. niger aggregate and A. carbonarius) isolated from wine grapes on agar medium with composition similar to that of grapes. They found that optimum temperatures for growth were between 30 and 37 °C, and optimum aW for growth was 0.98 in most cases. They also found differences among the different group of fungi studied: A. carbonarius growth rates were lower than that of A. niger aggregate and uniseriate isolated. Unfortunately, the authors did not discriminate the species among the A. niger aggregate used which made direct comparison with the present work more difficult. However, our results were similar for A. carbonarius. Marín et al. (1998) studied the effect of temperature (5– 45 °C), water activity (0.995–0.75) and their interactions on mycelial growth of one strain of A. niger isolated from maize grain on maize extract medium. They found optimum growth of A. niger at 0.994 and 37 °C and minimum at 0.90 and 15 °C. The range of aW and temperatures studied by these authors was wider than our ranges and the behaviour of A. niger in our conditions was quite similar. Mitchell et al. (2004) investigated the in vitro effect of water activity (0.85–0.987) and temperature (10–40 °C) on growth rates of six strains of A. carbonarius isolated from wine grapes. The optimum aW for growth varied from 0.93 to 0.987 depending on the strain, with the widest aW for growth at 25– 30 °C. This confirmed that optimum growth for A. carbonarius was at higher temperatures (25–35 °C) and intermediate aW level (0.97–0.985). This is the first report on the effect of environmental parameters on growth and lag phase prior to growth for A. awamori. In the present study, growth rates and lag phase prior to growth over a range of environmental conditions provide important information as it could assist in predicting the
possible fungal contamination of several substrates. Furthermore, the information is relevant since these species are isolated in high frequency on numerous substrates for human and animal consumption in Argentina. Further studies considering the effect of the environmental factors on growth and OTA production on natural substrate (peanut seeds, maize kernels, dried grapes and coffee beans) are in progress. Acknowledgements The authors are grateful to the Secretaría de Ciencia y Técnica, Universidad Nacional de Río Cuarto (SECyT) Res.; FONCyT-PICT, which supported this study through grants. Mic. Andrea Astoreca is grateful to Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) for a doctoral fellowship. References Abarca, M.L., Accensi, F., Bragulat, M.R., Cabañes, F.J., 2001. Current importance of ochratoxin A-producing Aspergillus spp. Journal of Food Protection 64, 903–906. Abarca, M.L., Accensi, F., Cano, J., Cabañes, F.J., 2004. Taxonomy and significance of black aspergilli. Antonie van Leeuwenhoek 86, 33–49. Bellí, N., Marín, S., Sanchis, V., Ramos, A.J., 2004. Influence of water activity and temperature on growth of isolates of Aspergillus section Nigri obtained from grapes. International Journal of Food Microbiology 96, 19–27. Dalcero, A., Magnoli, C., Hallak, C., Chiacchiera, S.M., Palacio, G., Da Rocha Rosa, C.A., 2002. Detection of ochratoxin A in animal feeds and capacity to produce this mycotoxin by Aspergillus section Nigri in Argentina. Food Additives and Contaminants 19, 1065–1072. IARC. International Agency for Research on Cancer, 1993. In: Ochratoxin, A. (Ed.), En: Monographs on the Evaluation of Carcinogenic Risks to Human: Some Naturally Occurring Substances; Food Items and Constituents, Heterocyclic Aromatic Amines and Mycotoxins, vol. 56. Lyon, France, pp. 489–521. Magan, N., Olsen, M. (Eds.), 2004. Mycotoxins in Food: Detection and Control, cap. 13. Woodhead Publishing Limited, Cambridge England, pp. 307–339. Magnoli, C., Violante, M., Combina, M., Palacio, G., Dalcero, A., 2003. Mycoflora and ochratoxin-producing strains of Aspergillus section Nigriin wine grapes in Argentina. Letters in Applied Microbiology 37, 179–184. Magnoli, C., Astoreca, A., Ponsone, L., Combina, M., Palacio, G., Da Rocha Rosa, C.A., Dalcero, A., 2004. Survey of mycoflora and ochratoxin A in dried vine fruits from Argentina markets. Letters in Applied Microbiology 39, 326–331. Magnoli, C., Hallak, C., Chiacchiera, S., Dalcero, A., 2006. Occurrence of ochratoxin A-producing fungi in commercial corn kernels in Argentina. Mycophatologia 161, 53–58. Marín, S., Sanchis, Sáenz, V.R., Ramos, A.J., Vinas, I., Magan, N., 1997. Ecological determinants for germination and growth of some Aspergillusand Penicillium spp. from maize grain. Journal of Applied Microbiology 84, 25–36. Marín, S., Sanchis, V., Ramos, A.J., Vinas, I., Magan, N., 1998. Environmental factors, in vitro interactions, and niche overlap between Fusarium moniliforme, F. proliferatum and F. graminearum, Aspergillus and Penicillium species from maize grain. Mycological Research 102, 831–837. Mitchell, D., Parra, R., Aldred, D., Magan, N., 2004. Water and temperature relations of growth and ochratoxin A production by Aspergillus carbonarius strains from grapes in Europe and Israel. Journal of Applied Microbiology 97, 439–445. Pardo, E., Marín, S., Sanchis, V., Ramos, A.J., 2005. Impact of relative humidity and temperature on visible fungal growth and OTA production of ochratoxigenic Aspergillus ochraceusisolates on grapes. Food Microbiology 22, 383–389. Pitt, J.I., Hocking, A.D., 1997. Fungi and Food Spoilage. Blackie Academic and Professional, London. Vol. II.
International Journal of Food Microbiology 119 (2007) 314 – 318 www.elsevier.com/locate/ijfoodmicro
Water activity and temperature effects on growth of Aspergillus niger, A. awamori and A. carbonarius isolated from different substrates in Argentina Andrea Astoreca a,⁎, Carina Magnoli a,c , María L. Ramirez a,c , Mariana Combina b,c , Ana Dalcero a,c a
Departamento de Microbiología e Inmunología, Facultad de Ciencias Exactas, Físico, Químicas y Naturales, Universidad Nacional de Río Cuarto, Ruta Nacional No. 36 Km 601, (5800) Río Cuarto, Córdoba, Argentina b Instituto Nacional de Tecnología Agropecuaria (INTA), Luján de Cuyo, Mendoza, Argentina c Member of the Research Career of CONICET, Argentina Received 28 May 2007; accepted 28 August 2007
Abstract The objectives of this study were to determine the effect of water activity, temperature, and their interactions on a) mycelial growth rate and b) the lag phase prior to grow of seven isolates of Aspergillus section Nigri isolated from peanuts, maize kernels, dried grapes and coffee cherries from Argentina. Three Aspergillus niger, three A. awamori and one A. carbonarius isolates examined showed optimum aW level for growth at 0.97 with optimal temperature of 30 °C. for most of the isolates and 25 °C for only one (A. awamori RCP176). Minimal aW for growth was 0.85 at the highest temperature tested. Overall growth was reduced up to 50% at 0.93 aW. Growth was also to a large extend inhibited at 0.85 aW for most isolates even after 21 days of incubation at temperatures lower than 30 °C. The analysis of variance of the effect of single (isolate, aW and temperature), two- and three-way interaction showed that all factors alone and all interactions were statistically significant (P b 0.001) in relation to growth rates and lag phase for A. niger, A. awamori and A. carbonarius isolates. These data are relevant since these species are isolated in high frequency on numerous substrates for human and animal consumption in Argentina. © 2007 Elsevier B.V. All rights reserved. Keywords: Aspergillus section Nigri; Fungal growth; Water activity; Temperature
1. Introduction Ochratoxin A (OTA) is receiving increasing attention worldwide because of the hazard it poses to human and animal health. The toxicological profile includes teratogenesis, nephrotoxicity and immunotoxicity. This toxin has been classified as a possible human renal carcinogen (group 2B) (International Agency for Research on Cancer, 1993). OTA is a toxin naturally produced by Aspergillus ochraceus and some species of Aspergillus section Nigri formerly A. niger group and A. carbonarius. These ochratoxigenic species have been reported in and upon a great variety of substrates from regions with warmer and tropical climate. They are able to tolerate diverse ⁎ Corresponding author. Tel.: +54 358 4676429; fax: +54 358 4676231. E-mail address: [email protected] (A. Astoreca). 0168-1605/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2007.08.027
conditions of moisture, pH and temperature (Pitt and Hocking, 1997; Abarca et al., 2001; Magan and Olsen, 2004). Ochratoxin A was until recently believed to be produced only by A. ochraceus and related species belonging to section Circumdati and by Penicillium verrucossum. However, some other Aspergillus has been reported as ochratoxigenic, numerous reports dealing with the production of OTA by members of Aspergillus section Nigri exist. In this section the reported OTA producing species are A. carbonarius and those now included in the so-called Aspergillus niger aggregate (i.e. A. awamori and A. niger) (Abarca et al., 2004). In Argentina, species included on Aspergillus section Nigri have been isolated in high frequency on wines grapes (Magnoli et al., 2003), dried vine fruits (Magnoli et al., 2004), corn kernels (Magnoli et al., 2006), peanut seeds and coffee cherries (personal communication). The ability of OTA production by
A. Astoreca et al. / International Journal of Food Microbiology 119 (2007) 314–318
these strains has also been demonstrated (Dalcero et al., 2002; Magnoli et al., 2003, 2004, 2006). Fungal growth results from the complex interaction of several factors (water availability, temperature and incubation time) and, therefore, an understanding of each factor involved is essential to understand the overall process and to predict fungal spoilage in agricultural and food products (Pardo et al., 2005). Little attention has been paid to the influence of incubation, temperature and changes of water activity either in media or in grains on fungal growth of A. niger and A. awamori. Marín et al. (1997) compared the effect of aW, temperature and their interaction on the germination rate, the lag phase and the mycelial growth profile of one isolate of A. niger on maize extract medium. Bellí et al. (2004) studied the effect of aW and temperature on mycelia growth of eight isolates of A. niger aggregate on a synthetic medium similar to grape composition. But, in the last study the authors did not discriminate which species among A. niger aggregate were used. The objectives of this study were to determine the effect of aW, temperature, and their interactions on a) mycelial growth
315
rate and b) lag phase prior to growth of seven isolates of Aspergillus section Nigri isolated from peanuts, maize kernels, dried grapes and coffee cherries from Argentina. 2. Materials and methods 2.1. Fungal species A total of seven isolates of Aspergillus section Nigri was used in this study. Two strains of A. niger (RCP42 and RCP176) isolated from peanut grains, two strains isolated from coffee beans: A. niger and A. awamori (RCC4 and RCC20, respectively), two strains of A. awamori isolated from maize kernels (RCM15 and RCM30) and one strain of A. carbonarius (RCDG30) isolated from dried grapes. These isolates were found to be OTA producers on YES medium (2% yeast extract, 15% sucrose) (Dalcero et al., 2002; Magnoli et al., 2003, 2004, 2006). The references in brackets are the codes of cultures held in the National University of Río Cuarto Collection.
▪
Fig. 1. Interaction of aW and temperature on the growth rate and lag phase of three isolates of A. niger, two on peanut extract medium (RCP42 and RCP176) and one on coffee extract medium (RCC4). Water activity levels: 0.85 (♦) 0.892 (□)—0.91(▴)—0.937(X)—0.955(⋄)—0.971( ) and 0.995 (▵).
316
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2.2. Medium The basic medium used in this study was a 3% (w/v) of each substrate (peanut, maize kernels, dried grapes and coffee beans) extract agar with a pH of 5.5. This was made by boiling 30 g of each ground substrate per litre in water for 45 min and filtering the resultant mixture through a double layer of muslin. The volume was made up to 1 l. The water activity of each basal medium was modified by the addition of known amounts of glycerol (Dallyn and Fox, 1980) to 0.85, 0.89, 0.91, 0.94, 0.95, 0.97 and 0.995. The water activity of representative samples of each medium was checked with an AquaLab Series 3 (Decagon Devices, Inc., WA, USA). Additional, control plates were prepared and measured at the end of the experiment in order to detect any significant deviation of the aW. 2.3. Inoculation and incubation Petri plates were inoculated centrally with a 4 mm diameter agar plug taken from the margin of a 7-day-old growing colony of each isolate on malt extract agar (MEA). Inoculated plates of the same aW were sealed in polyethylene bags. Triplicate sets of
each treatment were incubated at 15, 25 and 30 °C for 21 days. The experiment was repeated twice. Each isolate was only inoculated on the media, based on the substrate where it had originally been isolated. 2.4. Growth assessment Two perpendicular diameters of the growing colonies were measured daily until the colony reached the edge of the plate. The radii of the colonies were plotted against time, and a linear regression applied to obtain the growth rate (mm day− 1) as the slope of the line. Lag phase for growth was defined as the time (days) to reach 5 mm of diameter. 2.5. Statistical analysis In all cases, the linear regression of increase in radius against time (in days) was used to obtain the growth rates (mm day− 1) under each set of treatment conditions. The growth rates of each species were then evaluated by analysis of variance (ANOVA) using SigmaStat for Windows Version 2.03 (SPSS Inc.). When the analysis was statistically significant, the Tukey's multiple-
▪
Fig. 2. Effect of the interaction of aW and temperature on the growth rate and lag phase of three isolates of A. awamori, two on maize extract medium (RCM30 and RCM15) and one on coffee extract medium (RCC20). Water activity levels: 0.85 (♦) 0.892(□)—0.91(▴)—0.937(X)—0.955(⋄)—0.971( ) and 0.995 (▵).
A. Astoreca et al. / International Journal of Food Microbiology 119 (2007) 314–318
317
Fig. 3. Interaction of aW and temperature on the growth rate and lag phase of one isolate of A. carbonarius (RCDG30) on dried grapes extract medium.
comparison procedure test was used for separation of the means. Statistical significance was judged at the level P b 0.001. 3. Results Fig. 1 gives a diagrammatic representation of the interaction of aW and temperature on the growth rate (mm day− 1) and lag phase (h) of three isolates of A. niger, two on peanut extract medium (RCP42 and RCP176) and one on coffee extract medium (RCC4). The results showed that the maximum growth rates for the three isolates of A. niger were obtained at 0.97 aW and 30 °C. Temperature influenced the effect of aW on growth. The minimum aW for growth of the isolates of A. niger changed with temperature. At 0.85 aW – the lowest level of water stress tested – all the isolates were able to grow at 30 °C, although under this water availability the growth was very slow. When the temperature was 25 °C only one isolate (RCP42) was able to grow. At 15 °C, the growth rates of the isolates RCP42 and RCP176 were negligible when aW levels were low (0.85, 0.89, 0.91), increasing slightly at 0.94, 0.95, 0.97 and 0.995. A. niger RCC4 was unable to grow at any of the aW levels tested at 15 °C during the incubation period. Very short lag phases (b 24 h) occurred at 25–30 °C and 0.995 to 0.94. At lower temperatures the lag times increased
Table 1 Analysis of variance of effect of water (aW), temperature (T ), and different isolates (i), and their interactions on growth rate and lag phase of Aspergillus niger, A. awamori and A. carbonarius Source of variation
df
i aW T I × aW I×T aW × T I × aW × T
6 6 2 36 12 12 72
a
a
Growth rate MS
b
12.88 128.96 344.83 2.66 3.82 15.34 1.47
Degrees of freedom. b Mean square. c F-Snedecor. ⁎ Significant P b 0.001.
Lag phase F
c
111.60 ⁎ 1117.77 ⁎ 2988.90 ⁎ 23.06 ⁎ 33.15 ⁎ 132.94 ⁎ 12.74 ⁎
MS b
F
c
39339.53 839944.86 2972712.27 6836.45 31159.19 156942.20 7527.28
99.44 ⁎ 2123.22 ⁎ 7514.45 ⁎ 17.28 ⁎ 78.76 ⁎ 396.72 ⁎ 19.03 ⁎
considerably, especially at the low water activities assayed. Similar trends were found for all studied isolates. Fig. 2 shows the interaction effect of aW and temperature on the growth rate (mm day− 1) and lag phase (h) of three isolates of A. awamori, two on maize extract medium (RCM30 and RCM15) and one on coffee extract medium (RCC20). Aspergillus awamori strains RCM15 and RCM30 have an optimum aW to grow at 0.97 at 30 °C, whereas the optimum for the isolate RCC20 was 0.97 at 25 °C. The growth rates decreased as temperature changed from optimum to low marginal conditions. For the two isolates RCM30 and RCC20 and temperatures assayed, growth rates increased with aW, reaching the optimum at 0.97 aW and decreasing slightly at 0.995. The isolate RCM15 showed a similar behaviour at 30 °C but at 25 and 15 °C growth rates increased with aW, reaching the optimum at 0.995. At 15 °C, the growth of all the isolates was negligible when aWlevels were low (0.85, 0.89 and 0.91), increasing slightly at 0.94 to 0.995 aW. All the isolates were more tolerant to low aW at temperatures close to the optimum. The shortest lag phases for A. awamori were obtained at 0.995 to 0.94 aW at 30 °C. At marginal temperatures, the lag phases were markedly increased. Fig. 3 shows a diagrammatic representation of the interaction of aW and temperature on the growth rate (mm day− 1) and lag phase (h) of one isolate of A. carbonarius on dried grapes extract medium. The optimum aW for growth was 0.97 at all temperatures levels assayed, with the widest aW tolerance at 25–30 °C. The lag phase increased from b 24 h at 25–30 °C and 0.995–0.97 aW to N504 h at marginal temperatures and water availabilities. The analysis of variance of the effect of single (isolate, aW and temperature) two- and three-way interaction showed that all factors alone and all interactions were statistically significant (P b 0.001) in relation to growth rates and lag phase for A. niger , for A. awamori and A. carbonarius isolates (Table 1). 4. Discussion In the present study mycelial growth and lag phase prior to grow of seven isolates of Aspergillus section Nigri isolated from different substrates (peanut seeds, corn kernels, dried grapes and coffee beans) were found to be significantly
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influenced by aW, temperature and their interactions in vitro on agar based media. While the pattern of environmental factors effect was similar, the growth rates of the isolates varied significantly. Three A. niger, three A. awamori and one A. carbonarius isolates that were examined usually showed optimum aW level for growth at 0.97 with optimal temperature of 30 °C for most of the isolated and 25 °C for only one (A. awamori RCP176). Minimal aW for growth was 0.85 at the highest temperature tested. Overall growth was reduced up to 50% at 0.93 aW. Growth was also to a large extend inhibited at 0.85 aW for most isolates even after 21 days of incubation at temperatures lower than 30 °C. Lag times at the optimal temperature for A. niger, A. awamori and A. carbonarius isolates varied from 2 h at 0.97 aW to 260 h at 0.85 aW from 40 h at 0.97 aWto 235 h at 0.85 aW and from 2 h at 0.97 aW to 300 h at 0.85 aW, respectively, and increased by changing to marginal temperatures. Interspecies and intraspecies differences were found between the isolates, aW, temperature and their interactions. Few studies have examined the effect of different environmental conditions on the growth of Aspergillus section Nigri isolated from different substrates and most of these studies have been done on A. niger and A. carbonarius isolates. Bellí et al. (2004) studied the effects of water activity and temperature on growth of Aspergillus section Nigri (A. niger aggregate and A. carbonarius) isolated from wine grapes on agar medium with composition similar to that of grapes. They found that optimum temperatures for growth were between 30 and 37 °C, and optimum aW for growth was 0.98 in most cases. They also found differences among the different group of fungi studied: A. carbonarius growth rates were lower than that of A. niger aggregate and uniseriate isolated. Unfortunately, the authors did not discriminate the species among the A. niger aggregate used which made direct comparison with the present work more difficult. However, our results were similar for A. carbonarius. Marín et al. (1998) studied the effect of temperature (5– 45 °C), water activity (0.995–0.75) and their interactions on mycelial growth of one strain of A. niger isolated from maize grain on maize extract medium. They found optimum growth of A. niger at 0.994 and 37 °C and minimum at 0.90 and 15 °C. The range of aW and temperatures studied by these authors was wider than our ranges and the behaviour of A. niger in our conditions was quite similar. Mitchell et al. (2004) investigated the in vitro effect of water activity (0.85–0.987) and temperature (10–40 °C) on growth rates of six strains of A. carbonarius isolated from wine grapes. The optimum aW for growth varied from 0.93 to 0.987 depending on the strain, with the widest aW for growth at 25– 30 °C. This confirmed that optimum growth for A. carbonarius was at higher temperatures (25–35 °C) and intermediate aW level (0.97–0.985). This is the first report on the effect of environmental parameters on growth and lag phase prior to growth for A. awamori. In the present study, growth rates and lag phase prior to growth over a range of environmental conditions provide important information as it could assist in predicting the
possible fungal contamination of several substrates. Furthermore, the information is relevant since these species are isolated in high frequency on numerous substrates for human and animal consumption in Argentina. Further studies considering the effect of the environmental factors on growth and OTA production on natural substrate (peanut seeds, maize kernels, dried grapes and coffee beans) are in progress. Acknowledgements The authors are grateful to the Secretaría de Ciencia y Técnica, Universidad Nacional de Río Cuarto (SECyT) Res.; FONCyT-PICT, which supported this study through grants. Mic. Andrea Astoreca is grateful to Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) for a doctoral fellowship. References Abarca, M.L., Accensi, F., Bragulat, M.R., Cabañes, F.J., 2001. Current importance of ochratoxin A-producing Aspergillus spp. Journal of Food Protection 64, 903–906. Abarca, M.L., Accensi, F., Cano, J., Cabañes, F.J., 2004. Taxonomy and significance of black aspergilli. Antonie van Leeuwenhoek 86, 33–49. Bellí, N., Marín, S., Sanchis, V., Ramos, A.J., 2004. Influence of water activity and temperature on growth of isolates of Aspergillus section Nigri obtained from grapes. International Journal of Food Microbiology 96, 19–27. Dalcero, A., Magnoli, C., Hallak, C., Chiacchiera, S.M., Palacio, G., Da Rocha Rosa, C.A., 2002. Detection of ochratoxin A in animal feeds and capacity to produce this mycotoxin by Aspergillus section Nigri in Argentina. Food Additives and Contaminants 19, 1065–1072. IARC. International Agency for Research on Cancer, 1993. In: Ochratoxin, A. (Ed.), En: Monographs on the Evaluation of Carcinogenic Risks to Human: Some Naturally Occurring Substances; Food Items and Constituents, Heterocyclic Aromatic Amines and Mycotoxins, vol. 56. Lyon, France, pp. 489–521. Magan, N., Olsen, M. (Eds.), 2004. Mycotoxins in Food: Detection and Control, cap. 13. Woodhead Publishing Limited, Cambridge England, pp. 307–339. Magnoli, C., Violante, M., Combina, M., Palacio, G., Dalcero, A., 2003. Mycoflora and ochratoxin-producing strains of Aspergillus section Nigriin wine grapes in Argentina. Letters in Applied Microbiology 37, 179–184. Magnoli, C., Astoreca, A., Ponsone, L., Combina, M., Palacio, G., Da Rocha Rosa, C.A., Dalcero, A., 2004. Survey of mycoflora and ochratoxin A in dried vine fruits from Argentina markets. Letters in Applied Microbiology 39, 326–331. Magnoli, C., Hallak, C., Chiacchiera, S., Dalcero, A., 2006. Occurrence of ochratoxin A-producing fungi in commercial corn kernels in Argentina. Mycophatologia 161, 53–58. Marín, S., Sanchis, Sáenz, V.R., Ramos, A.J., Vinas, I., Magan, N., 1997. Ecological determinants for germination and growth of some Aspergillusand Penicillium spp. from maize grain. Journal of Applied Microbiology 84, 25–36. Marín, S., Sanchis, V., Ramos, A.J., Vinas, I., Magan, N., 1998. Environmental factors, in vitro interactions, and niche overlap between Fusarium moniliforme, F. proliferatum and F. graminearum, Aspergillus and Penicillium species from maize grain. Mycological Research 102, 831–837. Mitchell, D., Parra, R., Aldred, D., Magan, N., 2004. Water and temperature relations of growth and ochratoxin A production by Aspergillus carbonarius strains from grapes in Europe and Israel. Journal of Applied Microbiology 97, 439–445. Pardo, E., Marín, S., Sanchis, V., Ramos, A.J., 2005. Impact of relative humidity and temperature on visible fungal growth and OTA production of ochratoxigenic Aspergillus ochraceusisolates on grapes. Food Microbiology 22, 383–389. Pitt, J.I., Hocking, A.D., 1997. Fungi and Food Spoilage. Blackie Academic and Professional, London. Vol. II.