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Effect of Capsicum carotenoids on growth and ochratoxin A production by chilli and paprika Aspergillus spp. isolates
International Journal of Food Microbiology 142 (2010) 354–359
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International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o
Effect of Capsicum carotenoids on growth and ochratoxin A production by chilli and paprika Aspergillus spp. isolates L. Santos a, R. Kasper a, J. Gil-Serna b, S. Marín a, V. Sanchis a, A.J. Ramos a,⁎ a b
Food Technology Department, University of Lleida, XaRTA-UTPV, Av. Alcalde Rovira Roure 191, 25198 Lleida, Spain Department of Microbiology III, Faculty of Biology, University Complutense of Madrid, José Antonio Novais 2, 28040-Madrid, Spain
a r t i c l e
i n f o
Article history: Received 4 February 2010 Received in revised form 27 May 2010 Accepted 14 July 2010 Keywords: Ochratoxin A Aspergillus Growth Carotenoids
a b s t r a c t The aim of this study was to determine the effect of a natural carotenoid mixture (Capsantal FS-30-NT), containing capsanthin and capsorubin, on growth and mycotoxin production of ochratoxin A-producing A. ochraceus, A. westerdijkiae, and A. tubingensis isolates. One isolate of each species, previously isolated from paprika or chilli, was inoculated on Czapek Yeast extract Agar (CYA) medium supplemented with different amounts of capsantal (0 to 1%) and incubated at 10, 15 and 25 °C for 21 days. Growth rates and lag phases were obtained, and OTA production was determined at 7, 14 and 21 days. The taxonomically related A. ochraceus and A. westerdijkiae showed the same behavior at 15 °C, but A. ochraceus was able to grow at 10 °C and had higher growth rates at 25 °C. A. tubingensis had the highest growth rates and lowest OTA production capacity of the assayed isolates, and it was not able to grow at 10 °C. Capsantal addition resulted in increased lag phases at 15 °C for all the strains, while growth rates remained rather constant. At 25 °C capsantal reduced growth rates, with rather constant lag phases. However, the effect of capsantal on OTA production was inconclusive, because it depended on temperature or time, and mostly was not significant. Low temperature has been a crucial factor in OTA production, regardless of the capsantal concentration tested, especially for A. tubingensis and A. westerdijkiae. Industrial storage temperature for paprika and chilli is approximately 10 °C. If this temperature is maintained, mould growth and OTA production should be reduced. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Ochratoxin A (OTA) is a mycotoxin toxic to animals and man, mainly due to its nephrotoxic properties. The European Union has set strict limitations on OTA levels in various foodstuffs, such as cereals, coffee, raisins and wine (Commission Regulation EC No.1881/2006), and recently a new regulation for OTA maximum levels in spices (Capsicum powder included) has been set (Commission Regulation EU 105/2010). Aspergillus section Nigri and section Circumdati are notoriously known for their OTA production. The OTA-producers from section Circumdati are mainly strains of A. albertensis, A. alliaceus, A. ochraceus, A. melleus, A. ostianus, A. petrakii, A. sclerotiorum, A. sulfurous, A. wentii and A. westerdijkiae (Hesseltine et al., 1972; Varga et al., 1996; Frisvad et al., 2004), whereas the OTA-producers from section Nigri are A. awarori, A. carbonarius, A. foetidus, A. niger and A. tubingensis (Horie, 1995; Téren et al., 1996; Medina et al., 2005).
⁎ Corresponding author. Tel.: + 34 973702811; fax: + 34 973702596. E-mail address: [email protected] (A.J. Ramos). 0168-1605/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2010.07.018
Some Aspergillus species have been found in paprika and chilli samples: Aspergillus section Nigri, Aspergillus section Circumdati, A. flavus/parasiticus, A. nidulans, A. sclerotia, A. oryzae, A. terreus and A. versicolor, being A. flavus/parasiticus and A. section Nigri the most commonly found (Santos et al., 2008). Several studies reported OTA in paprika and chilli samples in a range between 0.4 and 281 μg/kg (ElKady et al., 1995; Patel et al., 1996; Thirumala-Devi et al., 2000; Fazekas et al. 2005; Goryacheva et al., 2006; Saha et al. 2007; Hierro et al. 2008; Santos et al., 2010). Paprika and chilli contain a large amount of carotenoids, including capsanthin, capsorubin, β-carotene, cryptoxanthin, and zeaxanthin. Another important carotenoid is capsaicin, responsible for the pungent characteristic of these products, which is only present in chilli peppers and has been shown to be effective against some bacteria and fungi (Cichewicz and Thorpe, 1996; Singh and Chittenden, 2008; Kraikruan et al., 2008; Tewksbury et al., 2008). However, chilli peppers, as well as paprika peppers, contain other components whose antimicrobial activities have not been wellstudied, as capsanthin or capsorubin. These two compounds, which are responsible for the red colour, can only be found in Capsicum products (Schweiggert et al., 2007).
L. Santos et al. / International Journal of Food Microbiology 142 (2010) 354–359
Some studies have demonstrated the effect of carotenoids in mycotoxigenic mould control, but they only have been assayed against a limited number of mould species (Masood et al., 1994; Norton, 1997). The aim of this study was to determine the effect of a natural carotenoid mixture, containing capsanthin and capsorubin, on growth and mycotoxin production of OTA-producing A. ochraceus, A. westerdijkiae, and A. tubingensis isolates. 2. Material and methods 2.1. Strains One OTA-producing isolate each of A. ochraceus (UdLTA 3.182), A. westerdijkiae (UdLTA 3.183), and A. tubingensis (UdLTA 3.178) was used. These moulds were previously isolated from paprika and chilli and belong to the Food Technology Department Collection of the University of Lleida, Spain. 2.2. Molecular identification of A. tubingensis, A. ochraceus and A. westerdijkiae In order to confirm the accuracy of morphological identification, the 3 isolates were subjected to molecular analysis with specific primers for these species. Genomic DNA of the isolates was obtained using DNeasy Plant Mini Kit (Qiagen, Valencia, Spain), according to the manufacturer's instructions. All genomic DNAs were tested for suitability for PCR amplification using universal primers ITS1 and ITS4 and the protocol described elsewhere (Henry et al., 2000). The specific PCR amplification protocol used to detect A. westerdijkiae and A. ochraceus include the primers set WESTF (5′-CTTCCTTAGGGGTGGCACAG-3′)/WESTR (5′-CAACCTGATGAAATAGATTGGTTG-3′) and OCRAF (5′-CTTTTTCTTTTAGGGGGCACAG-3′)/OCRAR (5′-CAACCTGGAAAAATAGTTGGTTG-3′), respectively, and the PCR protocols described in Gil-Serna et al. (2009). Specific PCR assay for A. niger/A. tubingensis was carried out using primers ITS1 (Henry et al., 2000) and NIG (5′ CCGGAGAGAGGGGACGGC 3′) and the PCR condition described in González-Salgado et al. (2005). In order to discriminate between A. niger and A. tubingensis, a RFLP analysis of the PCR product was performed. Aliquots of 10 μl of PCR amplification reactions obtained with ITS1/NIG primers were digested overnight with Rsa I (Afa I) as have been described in González-Salgado et al. (2005). All amplification reactions were carried out in volumes of 25 μL containing 3 μL (10–60 ng) of template DNA, 1.50 μL of each primer (20 μM), 2.5 μL of 10 × PCR buffer, 1 μL of MgCl2 (50 mM), 0.25 μL of dNTPs (100 mM) and 0.2 μL of Taq DNA polymerase (5 U/μL) supplied by the manufacturer (Ecogen, Barcelona, Spain). PCR products were detected in 2% agarose ethidium bromide gels in TAE 1 buffer (Tris– acetate 40 mM and EDTA 1.0 mM). The DNA ladder “Real escala no 2” (Durviz, Valencia, Spain) was used as molecular size marker.
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3.75 g; sucrose 22.5 g; agar 11.25 g; H2O 750 mL to 1 L) was used for mould cultures. Capsantal, dissolved in ethanol was assayed at the following concentrations: 0 g/L (control — CYA0%), 2.5 g/L (CYA0.25%), 5.0 g/L (CYA0.50%), 7.5 g/L (CYA0.75%), and 10 g/L (CYA1%), respectively. A control of CYA plus ethanol (CYA0%) and a control of only CYA were prepared. Caution must be taken in research with carotenoids such as capsanthin as they are susceptible to degradation when exposed to light and temperature higher than 60 °C. As a result, the medium was always manipulated under minimal exposure to light, especially direct light, during preparation. Capsantal was added when the medium temperature was lower than 60 °C before pouring. 2.5. Inoculation For inocula preparation, one plate of Malt Extract Agar (MEA) (Malt Extract 15 g; peptone 0.75 g; glucose 15 g; agar 9 g; H2O 750 mL to 1 L) per strain was inoculated and incubated for 7 days at 25 °C. On the seventh day, suspensions of 1–5 · 106 spores/mL (in sterile water plus Tween 80) for each strain were prepared, adjusted using a Thoma counting chamber. CYA plates were inoculated once in the centre, with an inoculation needle and exposed to minimal light for carotenoids protection, working under green light. 2.6. Growth conditions Three replicate plates of each capsantal concentration and controls per strain were incubated in the dark at 10, 15 and 25 °C. 2.7. Growth measurements Petri plates were examined daily, or as required, for 21 days and the diameter of the growing colonies were measured in two directions at right angles to each other. The increase in colony diameter was determined and used to calculate the growth rate (mm/day) under each set of treatment conditions for each strain. While the measurements were taken, plates were exposed to green light. 2.8. Determination of mould toxigenic capacity
Capsantal FS-30-NT (capsantal) is a source of red xanthophylls, obtained from red pepper powder and has a minimal of 30 g/kg of total carotenoids. Capsantal components include: red pepper extract, ethoxyquin and excipient. This mixture is composed of transcapsanthin, cis-capsanthin, β-carotene, trans-zeaxanthin, cryptoxanthin, capsorubin, trans-lutein, and other carotenoids. This product was purchased from ITPSA (Barcelona, Spain).
On days 7, 14, and 21 high performance liquid chromatography (HPLC) was used to determine the OTA production. The extraction method was a modification of the Bragulat et al. (2001) protocol. After incubation, 3 plugs (diameter 5 mm) were aseptically removed from the inner, middle, and outer area of each colony. The plugs were placed into 4 mL glass vials for extraction. Extraction was done adding 1 mL of methanol and using a vortex for 5 s. After 1 h, the vials were shaken again and the solvent was filtered (Millex HV filter 0.45 mm, Millipore, Bedford, MA, USA) on an HPLC glass vial. After filtration, 100 μL were injected in a HPLC system (Waters, Milford, MA, USA) with a reverse-phase C18 silica gel column (Waters Spherisorb® 5 μm ODS2 4.6 × 150 mm, Milford, MA, USA), kept in a column oven at 40 °C. Detection was achieved by fluorescence (Waters 2475 fluorescence detector, Waters, Milford, MA, USA); excitation and emission wavelengths were set at 333 and 460 nm, respectively. An isocratic mobile phase of acetonitrile:water: acetic acid (51:47:2, v/v/v) was used with a flow rate of 1.0 mL/min. LOD was 0.024 ppb and LOQ 0.072 ppb. Retention time of OTA was 5.5 min.
2.4. Medium
2.9. Statistical analysis
Czapek Yeast extract Agar (CYA) (KH 2 PO 4 0.75 g; Czapek concentrate 7.5 mL; trace metal solution 0.75 mL; yeast extract
Colony diameters (D) were plotted against time. For each treatment, the growth plots were fitted to the biphasic Baranyi's
2.3. Carotenoids
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L. Santos et al. / International Journal of Food Microbiology 142 (2010) 354–359
Fig. 1. Growth rates (μ, mm/d) and lag phases (λ, days) for A. ochraceus, A. tubingensis and A. westerdijkiae growing on CYA, CYA0%, CYA0.25%, CYA0.50%, CYA0.75% and CYA1% treatments at 10, 15 and 25 °C. Error bars indicate confidence intervals (95%).
function (1) (Baranyi and Roberts, 1994) by using Statgraphics Plus 5.1. (Manugistics, Inc, Maryland, USA). D=μ ×
1 t+ × logðexpð−μ × tÞ + expð−μ × λÞ−expð−μ × t−μ × λÞÞ μ
ð1Þ
OTA concentrations detected under each condition were also evaluated using a multifactor analysis of variance with capsantal concentration, temperature, strain, and time as factors. An ANOVA was also used to evaluate the effects of temperature in the control treatment (CYA). 3. Results
Growth parameters were estimated for each plate: maximum growth rate (μ, mm/day) and lag time (λ, days). A multifactor analysis of variance (ANOVA) was used to test the significance of the effects of the factors (capsantal concentration, temperature, and strain) and their interactions on the growth parameters (λ and μ) using JMP® 8.0.1 (SAS Institute Inc, Cary, NC, USA). The Tukey HSD test was used to differentiate among levels of factors.
3.1. Growth The non-linear regression model (simplified Baranyi's function) fitted well to our growth data against time for the different treatments. Whenever the diameter measurements were higher than 80 mm, no more readings were taken to prevent a false
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Table 1 Effects of strain, temperature, capsantal treatment and their different interactions on the responses of λ and μ. Source
Strain Temperature Treatment Strain × temperature Strain × treatment Temperature × treatment Strain × temperature × treatment
DF
2 2 5 4 10 10 20
Sum of squares
Prob N F
F ratio
λ
μ
λ
μ
λ and μ
350.6 6467.8 319.6 644.4 77.8 224.5 139.8
359.8 2120.7 73.5 693.1 25.3 99.7 30.1
515.3 9504.5 187.9 473.5 22.9 66.0 20.5
741.0 4367.3 60.6 713.7 10.4 41.0 6.2
b0.0001⁎ b0.0001⁎ b0.0001⁎ b0.0001⁎ b0.0001⁎ b0.0001⁎ b0.0001⁎
⁎ Significant P b 0.05.
stationary phase. The fitting was better at higher temperature, with R2 = 0.288–0.965 at 10 °C, R2 = 0.976–0.997 at 15 °C and R2 = 0.951– 0.997 at 25 °C. In Fig. 1, it is possible to observe μ and λ variation for all isolates at the assayed temperatures. All factors and all their interactions had a significant effect on both λ and μ (P b 0.001) (Table 1). According to the Tukey test, in the control (CYA), the isolate A. tubingensis grew faster than the other two isolates, A. ochraceus being the slowest (P b 0.05). At 10 °C, the A. westerdijkiae isolate started to grow earlier (7.68 days) but it grew slower (0.52 mm/day). At 15 °C, the A. tubingensis isolate started to grow earlier (1.67 days) and faster (3.49 mm/day), while A. ochraceus and A. westerdijkiae had similar values for λ and μ. At 25 °C, the A. ochraceus isolate started to grow earlier (0.43 days); the isolate of A. tubingensis grew faster (18.25 mm/day), while A. westerdijkiae and A. ochraceus grew at 8.97 and 7.58 mm/day, respectively. Regarding temperature, an increase from 10 to 15 °C, or from 15 to 25 °C, led to a λ decrease and a μ increase (P b 0.05). At 10 °C, growth was slow, and there was no observable effect of the different capsantal concentrations. No growth was observed for A. tubingensis and A. westerdijkiae strains, except on CYA control, while for the A. ochraceus strain growth occurred under all treatments, but there were no significant differences among them. At 15 °C, for A. tubingensis, the addition of capsantal at 0.50% led to a significantly decreased growth rate and increased lag phase, with no significant differences for the range 0.5–1.0%. The capsantal addition had no effect on the growth rate of A. westerdijkiae and A. ochraceus, although it increased the duration of the lag phase when it was used at 0.25–1.0%. At 25 °C, for A. tubingensis, growth was significantly inhibited by 0.5–1.0% capsantal addition, while no significant differences were found between controls and capsantal at 0.25%. Capsantal treatment in the range 0.75–1.0% led to a delay in the lag phase from 0.56 days in CYA to 1.71 days in CYA1%. For A. westerdijkiae and A. ochraceus the addition of capsantal at 0.25% or higher led to a significantly decreased growth rate, while no significant differences between lag phases of controls and treatments were found. 3.2. Toxigenic capacity No growth occurred before day 7 at 10 °C, thus no OTA analysis were carried out under this condition. All factors (strain, temperature, capsantal treatment and time) and all their interactions had a significant effect on the OTA production ability (P b 0.01) (Table 2). The A. westerdijkiae isolate had, on average, the highest OTA production ability on CYA control (36,381.13 ng OTA/g of agar), while there were no significant differences between the OTA production ability of A. ochraceus and A. tubingensis (5.36 and 5.03 ng OTA/g of agar, respectively). The A. westerdijkiae isolate exhibited the highest observed OTA production on day 14 at 15 °C (Fig. 2). The A. ochraceus isolate produced significantly higher OTA levels at 25 °C (mean 16.08 ng OTA/g of agar) compared to 10 °C and 15 °C. This isolate showed, on average, a higher OTA production at day 21
(21.43 ng OTA/g of agar). At 10 °C, capsantal treatment had no significant effect. At 15 °C, there were no significant differences between days 7 and 21 in OTA production ability, OTA production was lower at day 14 (3.02 ng OTA/g of agar); there was no significant effect of capsantal treatments, regardless of time. At 25 °C, increasing OTA production was observed from day 7 to day 21; on day 7 no significant differences between treatments were observed, while after 14 and 21 days significant increases in OTA production occurred with capsantal addition (from b5 ng/g to up to 70 ng OTA/g of agar). The A. tubingensis isolate produced similar levels of OTA at 15 and 25 °C and no OTA at 10 °C. Moreover, mean levels did not differ at 7, 14, or 21 days. At 15 °C, on day 7, OTA production occurred only in the controls, while no OTA was detected in the capsantal treatments; for days 14 and 21, there was a small increase in OTA production capacity with the presence of capsantal. At 25 °C, there were no significant differences between days 7 and 14, achieving on average a higher OTA production on day 21; capsantal enhanced OTA production (P b 0.05), being sometimes maximum at 1%. At 10 °C, the A. westerdijkiae isolate only produced a detectable level of OTA after 21 days and only in the CYA control. Moreover, mean levels were significantly higher at 15 than at 25 °C. At 15 °C, significant differences were not found between days 7 (27,704 ng OTA/g of agar) and 21, with the highest production at day 14 (110,021 ng OTA/g of agar); an increase of OTA production with capsantal concentration was observed on day 7 (from controls 341 ng OTA/g of agar to CYA0.75% 85,575 ng OTA/g of agar), while no significative differences due to capsantal were found after 14 and 21 days. At 25 °C, in general, OTA production decreased with time, however, no significant impact of capsantal treatment in OTA production was observed regardless of the incubation time. 4. Discussion Few studies have addressed the ecological requirements of the latest identified ochratoxigenic species in the Aspergillus sections Nigri and Circumdati. Until recently, A. ochraceus and A. westerdijkiae were considered to be the same species (A. ochraceus). Although these two species had the same behavior at 15 °C, A. ochraceus is more adapted to grow at 10 °C and has a higher growth rate at 25 °C than A. westerdijkiae. A. westerdijkiae is morphologically similar to A. ochraceus, though it is unable to grow at 37 °C (Frisvad et al., 2004). A. westerdijkiae mainly occurs in warmer climates, and thus, grew better at 25 °C (Magan and Aldred, 2006). Before the taxonomic separation of the two species A. ochraceus was described as a fungus able to grow between 8 °C and 37 °C, with an optimum at 24–31 °C and that could produce OTA between 12 and 37 °C with an optimum at 31 °C (ICMSF, 1996). In our study A. ochraceus had on average lower lag times than A. westerdijkiae although similar growth rates, whereas A. tubingensis had the higher growth rates and lower OTA production capacity. The A. westerdijkiae isolate was the major OTA producer. In previous studies, maximal OTA production was observed at high temperatures
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Fig. 2. OTA production capacity for A. ochraceus, A. tubingensis and A. westerdijkiae for CYA, CYA0%, CYA0.25%, CYA0.50%, CYA0.75% and CYA1% treatments at 10, 15 and 25 °C. Absence of bars indicates no growth.
(30–35 °C) for A. westerdijkiae and at 25–30 °C for A. ochraceus (Abdel-Hadi and Magan, 2009). In our study, the maximum OTA production was observed at 15 °C for A. westerdijkiae and 25 °C for A. ochraceus, although higher temperatures were not tested. Like our study, Selouane et al. (2009) observed that A. tubingensis could not grow at 10 °C. The same authors also observed that 25 °C was the optimum growth temperature for A. tubingensis, and its optimal temperature for OTA production was 30–37 °C (Selouane et al., 2009). In our study, although only three temperature levels were assayed, it was also observed that A. tubingensis grew better at 25 °C, but its maximum OTA production was observed at 15 °C. Usually, temperature ranges for OTA production are more restrictive than those for growth (Esteban et al., 2004). In other in vitro studies it was also observed that Aspergillus section Nigri has a lower OTA
production ability than Aspergillus section Circumdati (Bellí et al., 2004). There are only a few studies about the use of carotenoids in mould growth control. Norton (1997) observed that growth of A. flavus was usually not significantly affected by carotenoids. Conversely, Masood et al. (1994) observed that capsanthin had an inhibitory effect on the growth of A. flavus. In our study, capsantal addition resulted in increased lag phases at 15 °C for all the strains, while growth rates remained rather constant. However, at 25 °C the addition of capsantal resulted in reduced growth rates, with rather constant lag phases, thus the inhibitory effect of capsantal on the assayed isolates was confirmed. The effect of capsantal on OTA production was inconclusive, because it depended on temperature or time, and most of the time, it was not significant. There are only a few studies
L. Santos et al. / International Journal of Food Microbiology 142 (2010) 354–359 Table 2 Effects of strain, temperature, capsantal treatment, time and their different interactions on OTA production. Source
DF
Sum of squares
F ratio
Prob N F
Strain Temperature Time Treatment Strain × temperature Strain × time Temperature × time Strain × temperature × time Strain × treatment Temperature × treatment Strain × temperature × treatment Time × treatment Strain × time × treatment Temperature × time × treatment Strain × temperature × time × treatment
2 2 2 5 4 4 4 8 10 10 20 10 20 20 40
1.36E + 11 4.33E + 10 3.71E + 09 1.35E + 09 8.64E + 10 7.42E + 09 2.22E + 10 4.42E + 10 2.70E + 09 6.69E + 09 1.33E + 10 3.41E + 09 6.83E + 09 9.44E + 09 1.90E + 10
609.5 194.0 16.6 2.4 193.5 16.6 49.7 49.5 2.4 6.0 6.0 3.0 3.1 4.2 4.2
b 0.0001⁎ b 0.0001⁎ b 0.0001⁎ 0.0361⁎ b 0.0001⁎ b 0.0001⁎ b 0.0001⁎ b 0.0001⁎ 0.0087⁎ b 0.0001⁎ b 0.0001⁎ 0.0010⁎ b 0.0001⁎ b 0.0001⁎ b 0.0001⁎
⁎ Significant P b 0.05.
demonstrating the influence of carotenoids in toxin production, and none was performed with A. tubingensis, A. ochraceus or A. westerdijkiae. Norton (1997) observed that carotenoids can inhibit aflatoxin biosynthesis in A. flavus, and Masood et al. (1994) observed that capsanthin had the same effect on aflatoxin biosynthesis. As previously mentioned, the capsantal treatment had a significant effect on fungal growth, but its effect on OTA production (ng OTA per g of agar) is not clear. Some authors have suggested that production of OTA is not associated with rapid fungal growth; conversely higher growth rates appear to restrict OTA production (Häggblom, 1982). This study demonstrates that mould growth, and consequently OTA production capacity, can be influenced by intrinsic factors (substrate composition) and extrinsic factors (temperature and time). As observed, a low temperature has been a crucial factor in OTA production, regardless of the capsantal concentration tested, especially for A. tubingensis and A. westerdijkiae. The usual industrial storage temperature for paprika and chilli is approximately 10 °C; thus, if this temperature is maintained, mould growth and OTA production should be reduced. Acknowledgments The authors are grateful to the Spanish (Project AGL2007-66416C05-03) and Catalonian (XaRTA — Reference Network on Food Technology) Government for their financial support. References Abdel-Hadi, A., Magan, N., 2009. Influence of physiological factors on growth, sporulation and ochratoxin A/B production of the new Aspergillus ochraceus grouping. World Mycotoxin Journal 2 (4), 429–434. Baranyi, J., Roberts, T.A., 1994. A dynamic approach to predicting bacterial growth in food. International Journal of Food Microbiology 23, 277–294. Bellí, N., Pardo, E., Marín, S., Farré, G., Ramos, A.J., Sanchis, V., 2004. Occurrence of ochratoxin A and toxigenic potential of fungal isolates from Spanish grapes. Journal of the Science of Food and Agriculture 84, 541–546. Bragulat, M.R., Abarca, M.L., Cabañes, F.J., 2001. An easy screening method for fungi producing ochratoxin A in pure culture. International Journal of Food Microbiology 71, 139–144. Cichewicz, R.H., Thorpe, P.A., 1996. The antimicrobial properties of chile peppers (Capsicum species) and their uses in Mayan medicine. Journal of Ethnopharmacology 52, 61–70. European Commission, 2006. Commission Regulation (EC) No 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs. Official Journal of the European Union L 364, 5–24. European Commission, 2010. Commission Regulation (EU) No 105/2010 of 5 February 2010 amending Regulation (EC) No 1881/2006 setting maximum levels for certain contaminants in foodstuffs as regards ochratoxin A. Official Journal of the European Union L 35, 7–8. El-Kady, I.A., El-Maraghy, S.S.M., Mostafa, M.E., 1995. Natural occurrence of mycotoxins in different spices in Egypt. Folia Microbiologica 40, 297–300.
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Masood, A., Dogra, J.V.V., Jha, A.K., 1994. The influence of colouring and pungent agents of red Chilli (Capsicum annum) on growth and aflatoxin production by Aspergillus flavus. Letters in Applied Microbiology 18, 184–186. Medina, A., Mateo, R., López-Ocaña, L., Valle-Algarra, F.M., Jiménez, M., 2005. Study of Spanish grape mycobiota and ochratoxin A production by isolates of Aspergillus tubingensis and other members of Aspergillus section Nigri. Applied Environmetal Microbiology 71, 4696–4702. Norton, R.A., 1997. Effect of carotenoids on aflatoxins B1 synthesis by Aspergillus flavus. Phytopathology 87, 814–821. Patel, S., Hazel, C.M., Winterton, A.G.M., Mortby, E., 1996. Survey of ethnic foods for mycotoxins. Food Additives and Contaminants 13, 833–841. Saha, D., Acharya, D., Roy, D., Shrestha, D., Dhar, T.K., 2007. Simultaneous enzyme immunoassay for the screening of aflatoxins B1 and ochratoxin A in chili samples. Analytica Chimica Acta 584, 343–349. Santos, L., Marín, S., Sanchis, V., Ramos, A.J., 2008. Capsicum and mycotoxin contamination: state of the art in a global context. Food Science and Technology International 14, 5–20. Santos, L., Marín, S., Sanchis, V., Ramos, A.J., 2010. Co-occurrence of aflatoxins, ochratoxin A and zearalenone in Capsicum powder samples available on the Spanish market. Food Chemistry 122 (3), 826–830. Schweiggert, U., Kurz, C., Schieber, A., 2007. Effects of processing and storage on the stability of free and esterified carotenoids of red peppers (Capsicum annum L.) and hot chilli peppers (Capsicum frutescens L.). European Food Research and Technology 225, 261–270. Selouane, A., Bouya, D., Lebrihi, A., Decock, C., Bouseta, A., 2009. Impact of some environmental factors on growth and production of ochratoxin A of/by Aspergillus tubingensis, A. niger, and A. carbonarius isolated from Moroccan grapes. Journal of Microbiology 47 (4), 411–419. Singh, T., Chittenden, C., 2008. In-vitro antifungal activity of chilli extracts in combination with Lactobacillus casei against common sapstain fungi. International Biodeterioration & Biodegradation 62, 364–367. Téren, J., Varga, J., Hamari, Z., Rinyu, E., Kevei, F., 1996. Immunochemical detection of ochratoxin A in black Aspergillus strains. Mycopathologia 134, 171–176. Tewksbury, J.J., Reagan, K.M., Machnicki, N.J., Carlo, T.A., Haak, D.C., Peñaloza, A.L.C., Levey, D.J., 2008. Evolutionary ecology of pungency in wild chillies. Proceedings of the National Academy of Science of the United States of America (PNAS) 105 (33), 11808–11811. Thirumala-Devi, K., Mayo, M.A., Reddy, G., Reddy, S.V., Delfosse, P., Reddy, D.V.R., 2000. Production of polyclonal antibodies against ochratoxin A and its detection in chilies by ELISA. Journal of Agriculture and Food Chemistry 48, 5079–5082. Varga, J., Kevei, F., Rinyu, E., Teren, J., Kozakiewicz, Z., 1996. Ochratoxin production by Aspergillus species. 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International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o
Effect of Capsicum carotenoids on growth and ochratoxin A production by chilli and paprika Aspergillus spp. isolates L. Santos a, R. Kasper a, J. Gil-Serna b, S. Marín a, V. Sanchis a, A.J. Ramos a,⁎ a b
Food Technology Department, University of Lleida, XaRTA-UTPV, Av. Alcalde Rovira Roure 191, 25198 Lleida, Spain Department of Microbiology III, Faculty of Biology, University Complutense of Madrid, José Antonio Novais 2, 28040-Madrid, Spain
a r t i c l e
i n f o
Article history: Received 4 February 2010 Received in revised form 27 May 2010 Accepted 14 July 2010 Keywords: Ochratoxin A Aspergillus Growth Carotenoids
a b s t r a c t The aim of this study was to determine the effect of a natural carotenoid mixture (Capsantal FS-30-NT), containing capsanthin and capsorubin, on growth and mycotoxin production of ochratoxin A-producing A. ochraceus, A. westerdijkiae, and A. tubingensis isolates. One isolate of each species, previously isolated from paprika or chilli, was inoculated on Czapek Yeast extract Agar (CYA) medium supplemented with different amounts of capsantal (0 to 1%) and incubated at 10, 15 and 25 °C for 21 days. Growth rates and lag phases were obtained, and OTA production was determined at 7, 14 and 21 days. The taxonomically related A. ochraceus and A. westerdijkiae showed the same behavior at 15 °C, but A. ochraceus was able to grow at 10 °C and had higher growth rates at 25 °C. A. tubingensis had the highest growth rates and lowest OTA production capacity of the assayed isolates, and it was not able to grow at 10 °C. Capsantal addition resulted in increased lag phases at 15 °C for all the strains, while growth rates remained rather constant. At 25 °C capsantal reduced growth rates, with rather constant lag phases. However, the effect of capsantal on OTA production was inconclusive, because it depended on temperature or time, and mostly was not significant. Low temperature has been a crucial factor in OTA production, regardless of the capsantal concentration tested, especially for A. tubingensis and A. westerdijkiae. Industrial storage temperature for paprika and chilli is approximately 10 °C. If this temperature is maintained, mould growth and OTA production should be reduced. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Ochratoxin A (OTA) is a mycotoxin toxic to animals and man, mainly due to its nephrotoxic properties. The European Union has set strict limitations on OTA levels in various foodstuffs, such as cereals, coffee, raisins and wine (Commission Regulation EC No.1881/2006), and recently a new regulation for OTA maximum levels in spices (Capsicum powder included) has been set (Commission Regulation EU 105/2010). Aspergillus section Nigri and section Circumdati are notoriously known for their OTA production. The OTA-producers from section Circumdati are mainly strains of A. albertensis, A. alliaceus, A. ochraceus, A. melleus, A. ostianus, A. petrakii, A. sclerotiorum, A. sulfurous, A. wentii and A. westerdijkiae (Hesseltine et al., 1972; Varga et al., 1996; Frisvad et al., 2004), whereas the OTA-producers from section Nigri are A. awarori, A. carbonarius, A. foetidus, A. niger and A. tubingensis (Horie, 1995; Téren et al., 1996; Medina et al., 2005).
⁎ Corresponding author. Tel.: + 34 973702811; fax: + 34 973702596. E-mail address: [email protected] (A.J. Ramos). 0168-1605/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2010.07.018
Some Aspergillus species have been found in paprika and chilli samples: Aspergillus section Nigri, Aspergillus section Circumdati, A. flavus/parasiticus, A. nidulans, A. sclerotia, A. oryzae, A. terreus and A. versicolor, being A. flavus/parasiticus and A. section Nigri the most commonly found (Santos et al., 2008). Several studies reported OTA in paprika and chilli samples in a range between 0.4 and 281 μg/kg (ElKady et al., 1995; Patel et al., 1996; Thirumala-Devi et al., 2000; Fazekas et al. 2005; Goryacheva et al., 2006; Saha et al. 2007; Hierro et al. 2008; Santos et al., 2010). Paprika and chilli contain a large amount of carotenoids, including capsanthin, capsorubin, β-carotene, cryptoxanthin, and zeaxanthin. Another important carotenoid is capsaicin, responsible for the pungent characteristic of these products, which is only present in chilli peppers and has been shown to be effective against some bacteria and fungi (Cichewicz and Thorpe, 1996; Singh and Chittenden, 2008; Kraikruan et al., 2008; Tewksbury et al., 2008). However, chilli peppers, as well as paprika peppers, contain other components whose antimicrobial activities have not been wellstudied, as capsanthin or capsorubin. These two compounds, which are responsible for the red colour, can only be found in Capsicum products (Schweiggert et al., 2007).
L. Santos et al. / International Journal of Food Microbiology 142 (2010) 354–359
Some studies have demonstrated the effect of carotenoids in mycotoxigenic mould control, but they only have been assayed against a limited number of mould species (Masood et al., 1994; Norton, 1997). The aim of this study was to determine the effect of a natural carotenoid mixture, containing capsanthin and capsorubin, on growth and mycotoxin production of OTA-producing A. ochraceus, A. westerdijkiae, and A. tubingensis isolates. 2. Material and methods 2.1. Strains One OTA-producing isolate each of A. ochraceus (UdLTA 3.182), A. westerdijkiae (UdLTA 3.183), and A. tubingensis (UdLTA 3.178) was used. These moulds were previously isolated from paprika and chilli and belong to the Food Technology Department Collection of the University of Lleida, Spain. 2.2. Molecular identification of A. tubingensis, A. ochraceus and A. westerdijkiae In order to confirm the accuracy of morphological identification, the 3 isolates were subjected to molecular analysis with specific primers for these species. Genomic DNA of the isolates was obtained using DNeasy Plant Mini Kit (Qiagen, Valencia, Spain), according to the manufacturer's instructions. All genomic DNAs were tested for suitability for PCR amplification using universal primers ITS1 and ITS4 and the protocol described elsewhere (Henry et al., 2000). The specific PCR amplification protocol used to detect A. westerdijkiae and A. ochraceus include the primers set WESTF (5′-CTTCCTTAGGGGTGGCACAG-3′)/WESTR (5′-CAACCTGATGAAATAGATTGGTTG-3′) and OCRAF (5′-CTTTTTCTTTTAGGGGGCACAG-3′)/OCRAR (5′-CAACCTGGAAAAATAGTTGGTTG-3′), respectively, and the PCR protocols described in Gil-Serna et al. (2009). Specific PCR assay for A. niger/A. tubingensis was carried out using primers ITS1 (Henry et al., 2000) and NIG (5′ CCGGAGAGAGGGGACGGC 3′) and the PCR condition described in González-Salgado et al. (2005). In order to discriminate between A. niger and A. tubingensis, a RFLP analysis of the PCR product was performed. Aliquots of 10 μl of PCR amplification reactions obtained with ITS1/NIG primers were digested overnight with Rsa I (Afa I) as have been described in González-Salgado et al. (2005). All amplification reactions were carried out in volumes of 25 μL containing 3 μL (10–60 ng) of template DNA, 1.50 μL of each primer (20 μM), 2.5 μL of 10 × PCR buffer, 1 μL of MgCl2 (50 mM), 0.25 μL of dNTPs (100 mM) and 0.2 μL of Taq DNA polymerase (5 U/μL) supplied by the manufacturer (Ecogen, Barcelona, Spain). PCR products were detected in 2% agarose ethidium bromide gels in TAE 1 buffer (Tris– acetate 40 mM and EDTA 1.0 mM). The DNA ladder “Real escala no 2” (Durviz, Valencia, Spain) was used as molecular size marker.
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3.75 g; sucrose 22.5 g; agar 11.25 g; H2O 750 mL to 1 L) was used for mould cultures. Capsantal, dissolved in ethanol was assayed at the following concentrations: 0 g/L (control — CYA0%), 2.5 g/L (CYA0.25%), 5.0 g/L (CYA0.50%), 7.5 g/L (CYA0.75%), and 10 g/L (CYA1%), respectively. A control of CYA plus ethanol (CYA0%) and a control of only CYA were prepared. Caution must be taken in research with carotenoids such as capsanthin as they are susceptible to degradation when exposed to light and temperature higher than 60 °C. As a result, the medium was always manipulated under minimal exposure to light, especially direct light, during preparation. Capsantal was added when the medium temperature was lower than 60 °C before pouring. 2.5. Inoculation For inocula preparation, one plate of Malt Extract Agar (MEA) (Malt Extract 15 g; peptone 0.75 g; glucose 15 g; agar 9 g; H2O 750 mL to 1 L) per strain was inoculated and incubated for 7 days at 25 °C. On the seventh day, suspensions of 1–5 · 106 spores/mL (in sterile water plus Tween 80) for each strain were prepared, adjusted using a Thoma counting chamber. CYA plates were inoculated once in the centre, with an inoculation needle and exposed to minimal light for carotenoids protection, working under green light. 2.6. Growth conditions Three replicate plates of each capsantal concentration and controls per strain were incubated in the dark at 10, 15 and 25 °C. 2.7. Growth measurements Petri plates were examined daily, or as required, for 21 days and the diameter of the growing colonies were measured in two directions at right angles to each other. The increase in colony diameter was determined and used to calculate the growth rate (mm/day) under each set of treatment conditions for each strain. While the measurements were taken, plates were exposed to green light. 2.8. Determination of mould toxigenic capacity
Capsantal FS-30-NT (capsantal) is a source of red xanthophylls, obtained from red pepper powder and has a minimal of 30 g/kg of total carotenoids. Capsantal components include: red pepper extract, ethoxyquin and excipient. This mixture is composed of transcapsanthin, cis-capsanthin, β-carotene, trans-zeaxanthin, cryptoxanthin, capsorubin, trans-lutein, and other carotenoids. This product was purchased from ITPSA (Barcelona, Spain).
On days 7, 14, and 21 high performance liquid chromatography (HPLC) was used to determine the OTA production. The extraction method was a modification of the Bragulat et al. (2001) protocol. After incubation, 3 plugs (diameter 5 mm) were aseptically removed from the inner, middle, and outer area of each colony. The plugs were placed into 4 mL glass vials for extraction. Extraction was done adding 1 mL of methanol and using a vortex for 5 s. After 1 h, the vials were shaken again and the solvent was filtered (Millex HV filter 0.45 mm, Millipore, Bedford, MA, USA) on an HPLC glass vial. After filtration, 100 μL were injected in a HPLC system (Waters, Milford, MA, USA) with a reverse-phase C18 silica gel column (Waters Spherisorb® 5 μm ODS2 4.6 × 150 mm, Milford, MA, USA), kept in a column oven at 40 °C. Detection was achieved by fluorescence (Waters 2475 fluorescence detector, Waters, Milford, MA, USA); excitation and emission wavelengths were set at 333 and 460 nm, respectively. An isocratic mobile phase of acetonitrile:water: acetic acid (51:47:2, v/v/v) was used with a flow rate of 1.0 mL/min. LOD was 0.024 ppb and LOQ 0.072 ppb. Retention time of OTA was 5.5 min.
2.4. Medium
2.9. Statistical analysis
Czapek Yeast extract Agar (CYA) (KH 2 PO 4 0.75 g; Czapek concentrate 7.5 mL; trace metal solution 0.75 mL; yeast extract
Colony diameters (D) were plotted against time. For each treatment, the growth plots were fitted to the biphasic Baranyi's
2.3. Carotenoids
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Fig. 1. Growth rates (μ, mm/d) and lag phases (λ, days) for A. ochraceus, A. tubingensis and A. westerdijkiae growing on CYA, CYA0%, CYA0.25%, CYA0.50%, CYA0.75% and CYA1% treatments at 10, 15 and 25 °C. Error bars indicate confidence intervals (95%).
function (1) (Baranyi and Roberts, 1994) by using Statgraphics Plus 5.1. (Manugistics, Inc, Maryland, USA). D=μ ×
1 t+ × logðexpð−μ × tÞ + expð−μ × λÞ−expð−μ × t−μ × λÞÞ μ
ð1Þ
OTA concentrations detected under each condition were also evaluated using a multifactor analysis of variance with capsantal concentration, temperature, strain, and time as factors. An ANOVA was also used to evaluate the effects of temperature in the control treatment (CYA). 3. Results
Growth parameters were estimated for each plate: maximum growth rate (μ, mm/day) and lag time (λ, days). A multifactor analysis of variance (ANOVA) was used to test the significance of the effects of the factors (capsantal concentration, temperature, and strain) and their interactions on the growth parameters (λ and μ) using JMP® 8.0.1 (SAS Institute Inc, Cary, NC, USA). The Tukey HSD test was used to differentiate among levels of factors.
3.1. Growth The non-linear regression model (simplified Baranyi's function) fitted well to our growth data against time for the different treatments. Whenever the diameter measurements were higher than 80 mm, no more readings were taken to prevent a false
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Table 1 Effects of strain, temperature, capsantal treatment and their different interactions on the responses of λ and μ. Source
Strain Temperature Treatment Strain × temperature Strain × treatment Temperature × treatment Strain × temperature × treatment
DF
2 2 5 4 10 10 20
Sum of squares
Prob N F
F ratio
λ
μ
λ
μ
λ and μ
350.6 6467.8 319.6 644.4 77.8 224.5 139.8
359.8 2120.7 73.5 693.1 25.3 99.7 30.1
515.3 9504.5 187.9 473.5 22.9 66.0 20.5
741.0 4367.3 60.6 713.7 10.4 41.0 6.2
b0.0001⁎ b0.0001⁎ b0.0001⁎ b0.0001⁎ b0.0001⁎ b0.0001⁎ b0.0001⁎
⁎ Significant P b 0.05.
stationary phase. The fitting was better at higher temperature, with R2 = 0.288–0.965 at 10 °C, R2 = 0.976–0.997 at 15 °C and R2 = 0.951– 0.997 at 25 °C. In Fig. 1, it is possible to observe μ and λ variation for all isolates at the assayed temperatures. All factors and all their interactions had a significant effect on both λ and μ (P b 0.001) (Table 1). According to the Tukey test, in the control (CYA), the isolate A. tubingensis grew faster than the other two isolates, A. ochraceus being the slowest (P b 0.05). At 10 °C, the A. westerdijkiae isolate started to grow earlier (7.68 days) but it grew slower (0.52 mm/day). At 15 °C, the A. tubingensis isolate started to grow earlier (1.67 days) and faster (3.49 mm/day), while A. ochraceus and A. westerdijkiae had similar values for λ and μ. At 25 °C, the A. ochraceus isolate started to grow earlier (0.43 days); the isolate of A. tubingensis grew faster (18.25 mm/day), while A. westerdijkiae and A. ochraceus grew at 8.97 and 7.58 mm/day, respectively. Regarding temperature, an increase from 10 to 15 °C, or from 15 to 25 °C, led to a λ decrease and a μ increase (P b 0.05). At 10 °C, growth was slow, and there was no observable effect of the different capsantal concentrations. No growth was observed for A. tubingensis and A. westerdijkiae strains, except on CYA control, while for the A. ochraceus strain growth occurred under all treatments, but there were no significant differences among them. At 15 °C, for A. tubingensis, the addition of capsantal at 0.50% led to a significantly decreased growth rate and increased lag phase, with no significant differences for the range 0.5–1.0%. The capsantal addition had no effect on the growth rate of A. westerdijkiae and A. ochraceus, although it increased the duration of the lag phase when it was used at 0.25–1.0%. At 25 °C, for A. tubingensis, growth was significantly inhibited by 0.5–1.0% capsantal addition, while no significant differences were found between controls and capsantal at 0.25%. Capsantal treatment in the range 0.75–1.0% led to a delay in the lag phase from 0.56 days in CYA to 1.71 days in CYA1%. For A. westerdijkiae and A. ochraceus the addition of capsantal at 0.25% or higher led to a significantly decreased growth rate, while no significant differences between lag phases of controls and treatments were found. 3.2. Toxigenic capacity No growth occurred before day 7 at 10 °C, thus no OTA analysis were carried out under this condition. All factors (strain, temperature, capsantal treatment and time) and all their interactions had a significant effect on the OTA production ability (P b 0.01) (Table 2). The A. westerdijkiae isolate had, on average, the highest OTA production ability on CYA control (36,381.13 ng OTA/g of agar), while there were no significant differences between the OTA production ability of A. ochraceus and A. tubingensis (5.36 and 5.03 ng OTA/g of agar, respectively). The A. westerdijkiae isolate exhibited the highest observed OTA production on day 14 at 15 °C (Fig. 2). The A. ochraceus isolate produced significantly higher OTA levels at 25 °C (mean 16.08 ng OTA/g of agar) compared to 10 °C and 15 °C. This isolate showed, on average, a higher OTA production at day 21
(21.43 ng OTA/g of agar). At 10 °C, capsantal treatment had no significant effect. At 15 °C, there were no significant differences between days 7 and 21 in OTA production ability, OTA production was lower at day 14 (3.02 ng OTA/g of agar); there was no significant effect of capsantal treatments, regardless of time. At 25 °C, increasing OTA production was observed from day 7 to day 21; on day 7 no significant differences between treatments were observed, while after 14 and 21 days significant increases in OTA production occurred with capsantal addition (from b5 ng/g to up to 70 ng OTA/g of agar). The A. tubingensis isolate produced similar levels of OTA at 15 and 25 °C and no OTA at 10 °C. Moreover, mean levels did not differ at 7, 14, or 21 days. At 15 °C, on day 7, OTA production occurred only in the controls, while no OTA was detected in the capsantal treatments; for days 14 and 21, there was a small increase in OTA production capacity with the presence of capsantal. At 25 °C, there were no significant differences between days 7 and 14, achieving on average a higher OTA production on day 21; capsantal enhanced OTA production (P b 0.05), being sometimes maximum at 1%. At 10 °C, the A. westerdijkiae isolate only produced a detectable level of OTA after 21 days and only in the CYA control. Moreover, mean levels were significantly higher at 15 than at 25 °C. At 15 °C, significant differences were not found between days 7 (27,704 ng OTA/g of agar) and 21, with the highest production at day 14 (110,021 ng OTA/g of agar); an increase of OTA production with capsantal concentration was observed on day 7 (from controls 341 ng OTA/g of agar to CYA0.75% 85,575 ng OTA/g of agar), while no significative differences due to capsantal were found after 14 and 21 days. At 25 °C, in general, OTA production decreased with time, however, no significant impact of capsantal treatment in OTA production was observed regardless of the incubation time. 4. Discussion Few studies have addressed the ecological requirements of the latest identified ochratoxigenic species in the Aspergillus sections Nigri and Circumdati. Until recently, A. ochraceus and A. westerdijkiae were considered to be the same species (A. ochraceus). Although these two species had the same behavior at 15 °C, A. ochraceus is more adapted to grow at 10 °C and has a higher growth rate at 25 °C than A. westerdijkiae. A. westerdijkiae is morphologically similar to A. ochraceus, though it is unable to grow at 37 °C (Frisvad et al., 2004). A. westerdijkiae mainly occurs in warmer climates, and thus, grew better at 25 °C (Magan and Aldred, 2006). Before the taxonomic separation of the two species A. ochraceus was described as a fungus able to grow between 8 °C and 37 °C, with an optimum at 24–31 °C and that could produce OTA between 12 and 37 °C with an optimum at 31 °C (ICMSF, 1996). In our study A. ochraceus had on average lower lag times than A. westerdijkiae although similar growth rates, whereas A. tubingensis had the higher growth rates and lower OTA production capacity. The A. westerdijkiae isolate was the major OTA producer. In previous studies, maximal OTA production was observed at high temperatures
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Fig. 2. OTA production capacity for A. ochraceus, A. tubingensis and A. westerdijkiae for CYA, CYA0%, CYA0.25%, CYA0.50%, CYA0.75% and CYA1% treatments at 10, 15 and 25 °C. Absence of bars indicates no growth.
(30–35 °C) for A. westerdijkiae and at 25–30 °C for A. ochraceus (Abdel-Hadi and Magan, 2009). In our study, the maximum OTA production was observed at 15 °C for A. westerdijkiae and 25 °C for A. ochraceus, although higher temperatures were not tested. Like our study, Selouane et al. (2009) observed that A. tubingensis could not grow at 10 °C. The same authors also observed that 25 °C was the optimum growth temperature for A. tubingensis, and its optimal temperature for OTA production was 30–37 °C (Selouane et al., 2009). In our study, although only three temperature levels were assayed, it was also observed that A. tubingensis grew better at 25 °C, but its maximum OTA production was observed at 15 °C. Usually, temperature ranges for OTA production are more restrictive than those for growth (Esteban et al., 2004). In other in vitro studies it was also observed that Aspergillus section Nigri has a lower OTA
production ability than Aspergillus section Circumdati (Bellí et al., 2004). There are only a few studies about the use of carotenoids in mould growth control. Norton (1997) observed that growth of A. flavus was usually not significantly affected by carotenoids. Conversely, Masood et al. (1994) observed that capsanthin had an inhibitory effect on the growth of A. flavus. In our study, capsantal addition resulted in increased lag phases at 15 °C for all the strains, while growth rates remained rather constant. However, at 25 °C the addition of capsantal resulted in reduced growth rates, with rather constant lag phases, thus the inhibitory effect of capsantal on the assayed isolates was confirmed. The effect of capsantal on OTA production was inconclusive, because it depended on temperature or time, and most of the time, it was not significant. There are only a few studies
L. Santos et al. / International Journal of Food Microbiology 142 (2010) 354–359 Table 2 Effects of strain, temperature, capsantal treatment, time and their different interactions on OTA production. Source
DF
Sum of squares
F ratio
Prob N F
Strain Temperature Time Treatment Strain × temperature Strain × time Temperature × time Strain × temperature × time Strain × treatment Temperature × treatment Strain × temperature × treatment Time × treatment Strain × time × treatment Temperature × time × treatment Strain × temperature × time × treatment
2 2 2 5 4 4 4 8 10 10 20 10 20 20 40
1.36E + 11 4.33E + 10 3.71E + 09 1.35E + 09 8.64E + 10 7.42E + 09 2.22E + 10 4.42E + 10 2.70E + 09 6.69E + 09 1.33E + 10 3.41E + 09 6.83E + 09 9.44E + 09 1.90E + 10
609.5 194.0 16.6 2.4 193.5 16.6 49.7 49.5 2.4 6.0 6.0 3.0 3.1 4.2 4.2
b 0.0001⁎ b 0.0001⁎ b 0.0001⁎ 0.0361⁎ b 0.0001⁎ b 0.0001⁎ b 0.0001⁎ b 0.0001⁎ 0.0087⁎ b 0.0001⁎ b 0.0001⁎ 0.0010⁎ b 0.0001⁎ b 0.0001⁎ b 0.0001⁎
⁎ Significant P b 0.05.
demonstrating the influence of carotenoids in toxin production, and none was performed with A. tubingensis, A. ochraceus or A. westerdijkiae. Norton (1997) observed that carotenoids can inhibit aflatoxin biosynthesis in A. flavus, and Masood et al. (1994) observed that capsanthin had the same effect on aflatoxin biosynthesis. As previously mentioned, the capsantal treatment had a significant effect on fungal growth, but its effect on OTA production (ng OTA per g of agar) is not clear. Some authors have suggested that production of OTA is not associated with rapid fungal growth; conversely higher growth rates appear to restrict OTA production (Häggblom, 1982). This study demonstrates that mould growth, and consequently OTA production capacity, can be influenced by intrinsic factors (substrate composition) and extrinsic factors (temperature and time). As observed, a low temperature has been a crucial factor in OTA production, regardless of the capsantal concentration tested, especially for A. tubingensis and A. westerdijkiae. The usual industrial storage temperature for paprika and chilli is approximately 10 °C; thus, if this temperature is maintained, mould growth and OTA production should be reduced. Acknowledgments The authors are grateful to the Spanish (Project AGL2007-66416C05-03) and Catalonian (XaRTA — Reference Network on Food Technology) Government for their financial support. References Abdel-Hadi, A., Magan, N., 2009. Influence of physiological factors on growth, sporulation and ochratoxin A/B production of the new Aspergillus ochraceus grouping. World Mycotoxin Journal 2 (4), 429–434. Baranyi, J., Roberts, T.A., 1994. A dynamic approach to predicting bacterial growth in food. International Journal of Food Microbiology 23, 277–294. Bellí, N., Pardo, E., Marín, S., Farré, G., Ramos, A.J., Sanchis, V., 2004. Occurrence of ochratoxin A and toxigenic potential of fungal isolates from Spanish grapes. Journal of the Science of Food and Agriculture 84, 541–546. Bragulat, M.R., Abarca, M.L., Cabañes, F.J., 2001. An easy screening method for fungi producing ochratoxin A in pure culture. International Journal of Food Microbiology 71, 139–144. Cichewicz, R.H., Thorpe, P.A., 1996. The antimicrobial properties of chile peppers (Capsicum species) and their uses in Mayan medicine. Journal of Ethnopharmacology 52, 61–70. European Commission, 2006. Commission Regulation (EC) No 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs. Official Journal of the European Union L 364, 5–24. European Commission, 2010. Commission Regulation (EU) No 105/2010 of 5 February 2010 amending Regulation (EC) No 1881/2006 setting maximum levels for certain contaminants in foodstuffs as regards ochratoxin A. Official Journal of the European Union L 35, 7–8. El-Kady, I.A., El-Maraghy, S.S.M., Mostafa, M.E., 1995. Natural occurrence of mycotoxins in different spices in Egypt. Folia Microbiologica 40, 297–300.
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