Modelling Aspergillus flavus growth and aflatoxins production in pistachio nuts

doi:10.1016/j.fm.2012.07.018

Food Microbiology

Volume 32, Issue 2, December 2012, Pages 378-388

https://doi.org/10.1016/j.fm.2012.07.018 Get rights and content

Abstract

Aflatoxins (AFs) are the main contaminants in pistachio nuts. AFs production in pistachio has been attributed to Aspergillus flavus. The aim of this study was to apply existing models to predict growth and AFs production by an A. flavus isolated from pistachios as a function of moisture content and storage temperature of pistachios in order to test their usefulness and complementarities. A full factorial design was used: the moisture content levels assayed were 10, 15, 20, 25 and 30% and incubation temperatures were 10, 15, 20, 25, 30, 37 and 42 °C. Both kinetic and probability models were built to predict growth of the strain under the assayed conditions. Among the assayed models, cardinal ones gave a good quality fit for radial growth rate data. Moreover, the progressive approach, which was developed based on a reduced number of experimental points led to an improved prediction in the validation step. This is quite significant as may allow for improved experimental designs, less costly than full factorial ones. Probability model proved to be concordant in 91% of the calibration set observations. Even though the validation set included conditions around the growth/no-growth interface, there was a 100% agreement in the predictions from the data set (n = 16, cut off = 0.5) after 60 days. Similarly, the probability for AF presence was rightly predicted in 89% of the cases. According to our results EC maximum aflatoxin levels would be surpassed in a period as short as 1 month if pistachio nuts reach 20 °C, unless %mc is ≤10%.

Highlights

► The gamma concept allowed for simpler model designs for growth prediction. ► Unsafe aflatoxin levels can be reached in 1 month if pistachio nuts reach 20 °C, unless %mc is ≤10%. ► Probability models led to good prediction of growth and aflatoxin production by Aspergillus flavus. ► Probability would never reach 0.05 if pistachio %mc is 8% or storage temperature is 8 °C.

Introduction

Pistachio nuts are among the commodities with the highest risk of aflatoxins (AFs) contamination. In 2010, the Rapid Alert System for Food and Feed (RASFF) dealt with 640 notifications related to AFs. There was a significant reduction of notifications as regards AFs in nuts and nut products (168) compared to 2009 (283). This is at least partially related to the change in legislation whereby the maximum levels for AFs in almonds, hazelnuts and pistachios in EU legislation have been aligned with Codex Alimentarius maximum levels (RASFF, 2011).

The incidence of AFs contamination in tree nuts is low, but their levels are quite variable, and high levels develop in a small percentage of nuts (Schatzki, 1995). This is corroborated by the relative low number of positive samples shown in Table 1 (median level < limit of detection (LOD)), except for an Iranian study, where 95% of samples were positive for aflatoxin B1 (AFB1) with LOD = 0.1 μg/kg. In all cases AFB1 was the major detected aflatoxin. Moreover, maximum levels ranged from 0.37 μg/kg in the German study to 2200 μg/kg in a Swedish sample; this marked skewness in concentration distribution is definitely of concern.

In previous studies, Aspergillus accounted for 20% of total fungal contamination of pistachios from Iran, which showed mean contamination values of 1.6% (Khosravi et al., 2007). In California, Aspergillus species dominated the mycoflora of unsterilized pistachio nuts, with 28–56% of the total mycobiota being Aspergillus niger and 3–22% Aspergillus flavus; regarding surface-sterilized pistachio nuts, A. niger accounted for 36–44% and A. flavus for 0–7% (Bayman et al., 2002). Contamination of Algerian pistachio samples with A. flavus was as high as 58%; a total of 56.5% isolates of A. flavus tested for aflatoxin production were identified as aflatoxin-producing isolates (Fernane et al., 2010a). Similarly, in California, it has been demonstrated that 43% of the A. flavus isolates from pistachio orchards were potential aflatoxin producers when tested (Doster and Michailides, 1994). A. flavus contamination of pistachio samples taken in Spain was as high as 30%, but no Aspergillus parasiticus was found. From the 48 isolates of A. flavus tested for aflatoxin production, 34 (70.8%) were identified as aflatoxin producing isolates (Fernane et al., 2010b).

Moulds in the genus Aspergillus frequently decay the kernel of pistachio nuts prior to harvest when the nuts are still on the tree; but infection may also occur after harvesting, storage and transition (Bruce et al., 2003). Early splitting of the pistachio, immature nuts and the shell splitting in early growth period causes fungal contamination. Wind and insects can be other factors of fungal contamination in nut products. Concentration of aflatoxins prior or at maturity stage is generally low (Cheraghali and Yazdanpanah, 2010). It seems that early-split and hull cracked nuts that are not infected in the orchard may become infected during transport and handling. Considering a relative high incidence of fungal contamination of nuts, it seems that difference in climate conditions, methods of handling during harvesting (tearing of the hull, remaining on the ground for a extended period), drying process and transferring leading to mechanical damages of nuts and inadequate drying after rewetting for dehulling are determinant for the final aflatoxin content.

In addition, contamination can be due to the long-term storage, marketing under non-hygienic conditions including high moisture and temperature (Khosravi et al., 2007). High humidity and high temperature within bulk bins provide ideal conditions for the infection of early split and cracked nuts, which dramatically increases the incidence and level of aflatoxin contamination (Doster and Michailides, 1994). Therefore, postharvest handling of pistachio nuts is as important as determining the optimum harvest management for preventing aflatoxin contamination (Panahi and Khezri, 2011).

Pistachio is produced around the world in several warm arid climate countries like Iran, USA, Turkey, Syria and China, as major producers (FAOSTAT, faostat.fao.org/). In 2001, FAO published the Manual of the application of the HACCP system in mycotoxin prevention and control, considering two pistachio processing lines after harvest according to the different procedures applied in Asian producing countries. The fast dehulling process line involves fast dehulling (within 24 h after harvest) for preventing staining, floating segregation and quickly drying to 5–6% water content to prevent fungal development. The objective of this line is to reach a good-condition-for-storing product until it is further processed. This process is followed by the major producing countries. Other countries such Turkey or Syria, based on traditional practices, follow slow dehulling process lines, where pistachios are sun dried and stored for months until they are dehulled, segregated by either flotation and drying or by air gravity separators. Subsequent steps are followed by both lines, including sorting, roasting, packaging and storage/shipping. In contrast to many crops, tree nuts for export undergo minimal or very light processing, such as blanching, and the majority of the crop are consumed as whole nuts. There is thus little opportunity to reduce aflatoxin levels by artificial means and natural, consumer-acceptable methods, must be therefore found (Molyneux et al., 2007). Thus prevention of aflatoxigenic mould development in resting steps may be crucial. For prevention, a clear picture of the %mc/temperature conditions which are either conducive or not for growth and aflatoxin production by aflatoxigenic fungi is required. If preventive action cannot be achieved, corrective actions such as physical segregation need to be done.

The aim of this study was to apply existing models to predict growth and AFs production by an A. flavus isolated from pistachios as a function of moisture content and storage temperature of pistachios in order to test their usefulness and complementarities.

Section snippets

Pistachio nuts

Dehulled pistachios were purchased from a wholesaler in Lleida, Catalunya, Spain. Origin was Teheran, Iran, grade: 5 star; type: round.

Fungal isolates

An aflatoxigenic A. flavus isolate from pistachio was included in this research (UdL-TA 382). The reference in brackets is the code of the culture held in the Food Technology Department Culture Collection of University of Lleida, Spain.

Experimental design

A full factorial design was used in which two factors were assayed: moisture content (%mc) and temperature. The %mc levels

Primary model

Maximum radial growth rate (μ_R) and lag phase (λ) were estimated through Baranyi's primary model (Table 3). As four plates were taken at random for measurements the fitting was not as good as if one plate would have been measured along time. Growth kinetics of A. flavus followed, in general, a lag-linear function with no upper asymptote. No growth was observed at 10%mc regardless of the incubation temperature, as well as at 10 and 42 °C. At 15 °C growth only occurred at 20–30% mc.

Secondary model

Five different

Discussion

The first approach in this work was calculating the %mc/temperature levels which prevent growth. If growth is prevented during the storage and transfer steps no further aflatoxin accumulation will take place, thus this was the starting point. Among the assayed models, cardinal ones gave a good quality fit for radial growth rate data. An additional advantage of these models is the fact that they include biologically interpretable parameters, giving more insight into the behaviour of the strains (

Acknowledgements

The authors are grateful to Spanish government (project AGL2010-22182-C04-04) and EC, KBBE – Food, Agriculture and Fisheries and Biotechnology (project 222738- Selection and improving of fit-for-purpose sampling procedures for specific foods and risks), for their financial support.

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