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Biodegradation of ochratoxin A by Aspergillus section Nigri species isolated from French grapes: a potential means of ochratoxin A decontamination in grape juices and musts

Hend Bejaoui, Florence Mathieu, Patricia Taillandier, Ahmed Lebrihi
DOI: http://dx.doi.org/10.1111/j.1574-6968.2005.00073.x 203-208 First published online: 1 February 2006

Abstract

Ochratoxin A (OTA) is a very dangerous mycotoxin, the presence of which is often reported in different foods, as well as in beverages such as grapes, grape juices and wines. Detoxifying these products is therefore of prime importance in protecting consumer health, and biological approaches have been the most promising methods. In this report, 40 isolates representing the black apergilli species Aspergillus carbonarius, A. niger aggregate and A. japonicus, isolated on French grapes, were assessed for OTA degradation capacities in CZAPEK yeast extract broth (CYB) and in a synthetic grape juice medium (SGM) contaminated with OTA at 2 mg L−1 (5 μM). It was clearly observed that in both media these fungi had the ability to degrade OTA to OTα (ochratoxinα). However, there were differences between the media used and species tested during OTA degradation. In SGM and CYB, 77% and 45% of the isolates, respectively were able to degrade more than 80% of the OTA. Despite a better growth on SGM, specific OTA degradation was higher on CYB for most of the isolates. Kinetic studies carried out on SGM with three black Aspergillus isolates all showed different OTA degradation rates. After 9 days of incubation, OTα had decreased, whereas an unknown compound appeared. A. niger could be the first interesting species for OTA detoxification processes, followed by A. japonicus.

Keywords
  • detoxification
  • ochratoxin A
  • ochratoxin α
  • black aspergilli
  • grape juice
  • must

Introduction

Ochratoxin A (OTA) is a very dangerous fungal secondary metabolite exhibiting nephrotoxic, immunosuppressive, teratogenic and carcinogenic properties (Smith & Moss, 1985). It is produced by a number of ubiquitous moulds of Penicillium and Aspergillus genera that contaminate agricultural commodities, either before harvest or during storage (Moss, 1996; Galtier, 1999). Ochratoxin A has been detected in many different food products such as cereals (Jorgensen et al., 1996), coffee (Pittet et al., 1996), beer (Visconti et al., 2000), dried vine fruits (Mac Donald et al., 1999) grapes, grape juices and wines (Zimmerli & Dick, 1995).

According to the Codex Alimentarius Commission, 15% of the total intake of OTA is due to wine (Codex Alimentarius Commission, 1998), which is also considered as the second major source of OTA intake after cereals (Codex Alimentarius Commission, 1998). Moreover, grape juices were shown to contain more OTA than some wines and so to contribute to the OTA intake of children (Zimmerli & Dick, 1996). OTA contamination in grapes and grape products could therefore be considered a serious health problem. To understand the origin of this contamination, efforts have been made to control it in various countries, especially those in Europe (Ochra Wine Risk Project).

Prevention of OTA production and/or the detoxification of contaminated products is then of prime importance in protecting consumer health. Several physical and chemical methods have been proposed in order to remove mycotoxins (Sinha, 1998). Although some of these methods could be promising [ozone (McKenzie et al., 1997), alkaline hydrogen peroxide (Fouler et al., 1994), or gamma irradiation (Refai et al., 1996)], others are not recommended for practical decontamination [hypochlorite treatment (Castegnaro et al., 1991), ammoniation (Chelkowski et al., 1982), or heat treatment (Boudra et al., 1995)]. Biological approaches are now being widely studied (Sweeny & Dobson, 1998), but none of them have focused principally on the decontamination of grape juices.

According to studies in French vineyards, black aspergilli were the main microorganisms in grapes and which colonized berries from setting to harvest (Bejaoui et al., 2005). Our objective in this work was to carry out a large-scale screening of black aspergilli isolated from French grapes, in order to determine their capacity to degrade OTA in a laboratory medium (CYB) and for the first time in a simulated grape juice (SGM).

A study of the kinetics of OTA in SGM and of its degradation metabolites, as well as comparisons between the OTA degradation profiles of Aspergillus carbonarius, A. niger and A. japonicus, were achieved for the first time during this survey.

Materials and methods

Isolates

Forty fungi belonging to three species of black aspergilli (Aspergillus carbonarius, A. japonicus and A. niger) isolated on French grapes were used.

Culturing media

For the fungal isolates both a modified CZAPEK yeast extract broth medium (CYB) and a synthetic grape juice medium (SGM) were used.

The synthetic grape juice (SGM) was prepared by dissolving 70 g glucose d(+) (Fisher Bioblock Scientific, Illbirch, France), 30 g fructose d(−) (Sigma, Saint Quentin, Fallavier, France), 7 g tartaric acid l(−) (Sigma), 10 g malic acid l(−) (VWR International, Fontenay sous Bois, France), 0.67 g KH2PO4 (SUBRA, Toulouse, France), 1.5 g MgSO4, 7H2O (SUBRRA), 0.15 g NaCl (Fisher), 0.15 g CaCl2 (SUBRA), 0.0015 g CuCl2, 0.021 g FeSO4, 7H2O (SUBRA), 0.0075 g ZnSO4, 7H2O (Fisher) and 0.05 g hydrated catechin (Sigma) in 1 L distilled water and its pH was adjusted to 3.8–4.0 with 2 N KOH.

A modified CZAPEK yeast extract broth medium (CYB) was prepared by dissolving 30 g saccharose, 5 g yeast extract, 50 mL solution A [(for 500 mL) (20 g NaNO3, 5 g KCl, 5 g MgSO4.7H2O, 0.1 g FeSO4·7H2O)], 50 mL solution B [(for 500 mL): 10 g, K2HPO4], 1 mL solution C [(100 mL), 1 g ZnSO4·7H2O, 0.5 g CuSO4·5H2O)] in 1 L of distilled water.

These laboratory media were supplemented with ochratoxin A at a concentration of 2 mg L−1 (5 μM).

Fermentation conditions

The measurement of OTA degradation capacity was undertaken in 2 mL of the appropriate medium (CYB or SGM), under agitation (240 r.p.m.), at 25°C for 12 days. Each medium was inoculated at 1% from a conidial suspension (107 conidia mL−1). Their final dry weights were determined using dried mycelium after centrifugation and were expressed in g L−1. A specific OTA degradation capacity was calculated by dividing the total amount of degraded OTA by the final dry weight (mg g−1).

Kinetic studies of OTA degradation and OTα production for the three Aspergillus isolates (A. carbonarius SA332, A. japonicus AX35 and A. niger GX312) were conducted during a batch fermentation in 20 mL of SGM media in Erlenmeyer flasks, at 25°C under agitation at 240 r.p.m.

A negative control (control 1) – an OTA contaminated medium without fungi – was used to calculate the OTA removal percentage. A positive control (control 2) – a fungal culture in a medium without OTA – was also used.

All assays were performed in triplicate.

OTA extraction

For all samples, after removal of the fungal mycelium, 1 mL of supernatant was extracted twice with ethyl acetate [volume in volume (v/v)], and then evaporated until dry before dissolving in 0.5 mL of methanol for the HPLC analysis.

Detection and quantification of OTA and OTα

Ochratoxin A and OTα were detected and quantified by reversed-phase high-performance liquid chromatography (HPLC). The HPLC apparatus consisted of a solvent delivery system and fluorescence (λex=332 nm; λem=466 nm) and UV detectors. The analytical column was a 150 × 4.6 mM Uptisphere 5 μm C18 ODB (AIT, France) fitted with a guard column of 10 × 4 mm. The mobile phase consisted of a mixture of HPLC grade acetonitrile : water : acetic acid (100 : 99.8 : 0.2) at a flow rate of 1 mL min−1 and the column temperature was at 30°C. The data acquisition system was a Kroma 3000 (BIO-TEK, Milan, Italy). Injections were made with an autoinjector (BIO-TEK) and the injection volume 80 μL. Ochratoxin A was identified by its retention time (33 min) according to a standard from Sigma (Steinheim, Germany). OTα was identified at 17 min according to a standard prepared by the total degradation of OTA by carboxypeptidase A (EC3.4.17.1) from bovine pancreas (Sigma, type II-PMSF). OTA and OTα were quantified by measuring the peak area and using standard solutions.

The percentage of OTA degradation was calculated according to the following equation: 100 × [1−(peak area of OTA/peak area of OTA in control 1)].

Result

Screening of Aspergillus sectionNigri isolates for OTA detoxifying activity

Forty Aspergillus section Nigri isolates (A. carbonarius, A. niger and A. japonicus) were tested for their ability to degrade OTA in both CYB and SGM. The A. carbonarius isolates were chosen due to their low OTA production, and A. niger and A. japonicus isolates because they were not ochratoxigenic. All were cultured on both media (CYB and SGM), which were initially contaminated with OTA (2 mg L−1), and all showed significant capacities to degrade the mycotoxin after 12 days of incubation. For all these species, OTα, an OTA degradation metabolite, was produced (data not shown). Figure 1 shows the percentage of OTA degradation for all isolates on both media. OTA degradation occurred more frequently in SGM than in CYB, with 77% and 45%, respectively of the isolates able to degrade more than 80% of the OTA. In SGM, all the isolates degraded at least 30% of the OTA, whereas in CYB some isolates could not decrease the initial concentration of mycotoxin at all. A. niger was the best at degrading OTA, followed by A. japonicus and A. carbonarius. Nevertheless, fungal growth was quite different on the two media. Specific OTA degradation was calculated and is shown in Fig. 2. Despite a better growth on SGM, specific OTA degradation was greater on CYB for most of the isolates.

Figure 1

Ochratoxin A (OTA) removal (%) by the 40 isolates of black aspergilli species (Aspergillus carbonarius, A. niger and A. japonicus) in CZAPEK yeast extract broth (CYB) and in a synthetic grape juice medium (SGM) initially contaminated at 2 mg L−1 OTA.

Figure 2

Specific ochratoxin A (OTA) degradation activity (mg g−1) of 40 isolates of black aspergilli (Aspergillus carbonarius, A. niger and A. japonicus) in CZAPEK yeast extract broth (CYB) and in a synthetic grape juice medium (SGM) initially contaminated at 2 mg L−1 OTA.

Kinetics of Ochratoxin A degradation by three isolates of Aspergillus section Nigri species on SGM

The kinetics of OTA degradation by one isolate from each arbitrarily chosen species: A. carbonarius SA332, A. niger GX312 and A. japonicus AX35, were examined for OTA (5 μM) in an SGM (Fig. 3).

Figure 3

Kinetics of ochratoxin A (OTA) and OTα in the presence of Aspergillus carbonarius, A. niger and A. japonicus on a synthetic grape juice medium (SGM) containing an initial concentration of 5 μM OTA.

Regardless of the species tested, OTA was degraded and hydrolysed to OTα. After 9 days of incubation, the amounts of OTα decreased and an unknown compound appeared (data not shown). Particularities in the OTA kinetics for each species are noteworthy. During the first 3 days of incubation, A. niger degraded OTA relatively slowly (20%) compared to A. carbonarius (55%) and A. japonicus (80%). However, after the third day, OTA degradation by A. niger became the most rapid and reached 99% after 5 days. By this time, A. carbonarius and A. japonicus had reduced the levels of OTA by 83% and 89%, respectively.

Discussion

In this study, we examined the OTA degrading activities of 40 isolates of black aspergilli on two media, CYB and SGM. Removal of OTA was observed for almost all black aspergilli isolates and degradation levels reached 98–99% in some cases.

Regardless of the species, OTA was hydrolysed to OTα, which has lower toxicity (Harwig, 1974). This OTA metabolism has previously been observed for different microorganisms like protozoa (in the gastrointestinal tracts of ruminants) (Galtier & Alvinerie, 1976; Hult et al., 1976), the bacteria Acinetobacter calcoaceticus (Hwang & Draughon, 1994), Phenylobacter immobile (Wegst & Lingens, 1983), and fungi A. niger (Xiao et al., 1996; Varga et al., 2000), as well as black aspergilli isolated from Portuguese grapes (Abrunhosa et al., 2002). Differences were observed between the OTA degradation capacities of the different species and for the same isolate, depending on the medium. That a higher OTA degradation was always obtained in SGM than in CYB can be attributed to the better fungal growth in SGM. A carboxypeptidase, which was previously found to be able to hydrolyse OTA to OTα (Deberghes et al., 1995), may be involved in OTA degradation. However, no data have been found in the literature regarding the presence of this enzyme in aspergilli. To explain the higher specific degradation activity observed in CYB, two hypotheses could be proposed. First, a greater quantity of enzyme could be synthesized by fungal cells in CYB. Second, in the case of an extracellular enzyme, this medium is more favourable for greater activity.

In view of the potential applications of Aspergillus sp. in the biological decontamination of grape juice, kinetics studies were done in SGM. For the three species, OTA was totally degraded to OTα, but following different performance profiles. Despite the initial lag phase, A. niger was the quicker species: in 5 days 99% of the OTA had been degraded. This species could be useful where total OTA degradation is required. In cases where a partial degradation was desired, A. carbonarius or A. japonicus could be used to reduce OTA concentrations to below maximum level of tolerance in wine (2 μg L−1) fixed by EU legislation.

The three species of black aspergilli were then able to degrade OTα to an unknown compound that could probably result from a modification of its isocoumarin ring, as previously reported by Galtier & Alvinerie (1976). However, this pathway is still unknown.

Although Hwang & Draughon (1994) found that A. niger was unable to degrade OTA, Varga (2000) and Abrhunosa (2002) found isolates among these species that were able to decrease OTA concentrations in initially contaminated media. These latter results are consistent with ours: in both studies all isolates were obtained from grapes. Furthermore, A. niger is one of the few fungi which has received GRAS (generally recognized as safe) status from the US Food and Drug Administration (due to its low toxicity) and could therefore be of interest in further uses for the biological elimination of OTA in grape juices and musts. However, A. japonicus, which is also frequently used in fungal biotechnology could be of interest as it has only twice been reported as ochratoxigenic (Dalcero et al., 2002; Battilani et al., 2003). Our isolate exhibited a potential degradation capacity as notable as A. niger.

Acknowledgements

This work was supported by grants from the European Union (QLK1-CT-2001-01761) and the French ‘Ministère de la jeunesse de l'éducation et de la recherche’ (AQS no. 02 PO571).

References

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