Image-Based Screening for the Identification of Bright Greenish Yellow Fluorescence on Pistachio Nuts and Cashews

The development of screening methodologies for a rapid identification of crops contaminated with aflatoxin is of great interest to agro-food industry. The objective of this work was to develop an image algorithm able to identify bright greenish yellow fluorescence (BGYF) on pistachio nuts and cashews. Previous researchers established that the presence of BGYF indicates that there is a high probability of aflatoxin contamination. Since BGYF is not a definitive indicator of aflatoxin contamination, samples emitting fluorescence should be removed and tested for aflatoxins by chemical means. This study, conducted in a static way, is an important step towards the development of a new more accurate and automatic aflatoxin screening method based on a vision system. In this work, a total of 352 samples of pistachio nuts and cashews were evaluated, half of which came from lots contaminated with aflatoxin. Two images in the 410–600 nm optical range were acquired for each sample. Imaging algorithms were developed to identify samples with fluorescent stains caused by BGYF. According to the image analysis results, nut samples were classified into two groups: fluorescent stains (FS) and non-fluorescent stains. Both BGYF and non-fluorescent samples were analyzed for aflatoxin. The laboratory analysis results showed a high correlation with the camera classification: pistachios and cashews placed in the FS group by the vision system contained 92 % and 82 % of the total number of nuts contaminated with aflatoxin, respectively. Moreover, a discriminant analysis of reflectance data was carried out in order to select the optimal optical range to detect BGYF, both in pistachio nuts, i.e., 480 and 520 nm, and in cashews, i.e., 440 and 600 nm.     

X-ray imaging for fungal necrotic spot detection in pistachio nuts

The potential of X-ray imaging for detecting pistachios with kernel necrotic (KN) spots was the main objective of this study. X-ray images of whole pistachios were compared with colour pictures of the same nuts taken after the kernel was opened by splitting it in two. The necrotic spots appear in the X-ray images as darker gray areas of almost round shape. Also, pistachios with KN spots contained about 60 times more aflatoxin than healthy ones in an aflatoxin contaminated sample. Elimination of such nuts will reduce aflatoxin in contaminated batches and improve the quality in batches free of aflatoxin.     

Mathematical approach for sampling plan performance assessment for aflatoxin B1 in pistachios

According to the EU Regulation 165/2010, the level of aflatoxin B1 and total aflatoxins in pistachios must not exceed 8 μg/kg and 10 μg/kg, respectively. Due to the heterogeneous distribution of this contaminant, the performance of sampling plans highly depends on the way they are designed. So, the sampling plan to be used must be assessed thanks to an OC (Operating Characteristic) curve showing both consumer and producer risks. The first risk is the risk of authorizing pistachios for sale while the contamination level is above the threshold; whereas the second risk is the risk of rejecting a lot having a contamination level under the threshold.The method developed the use of early split contamination levels and incidence in individual pistachios to derive OC curves for various sampling plan designs for aflatoxin B1. Contamination levels in individuals were calculated from small sample contamination levels, thus having a negligible probability of containing more than one contaminated individual. Incidence levels of early split pistachios were derived for each harvest day. Indeed, early split nuts are dubious nuts that were considered to contain all the aflatoxin content found in a global sample. As their percentage increased along the harvesting period, it led to various contamination distributions in 10 kg samples. The EU sampling plan (Regulation 178/2010) was found to give a consumer risk with a probability of acceptance at 5% for a lot mean aflatoxin B1 concentration of 75.34 μg/kg and a producer risk with a probability of acceptance at 95% for a lot mean concentration of 1.62 μg/kg. Finally, the performance of several sampling plan designs was evaluated to demonstrate how to manipulate the number of samples to reduce misclassification of pistachio lots.     

Mycoflora and aflatoxin contamination in shelled pistachio nuts

A total of 143 pistachio nut samples collected during harvest, storage and processing were examined for mould growth and aflatoxin production. The mould count was in the range of 10³?10⁴ cfu g^?1 and 10⁵?10⁶ cfu g^?1 for the harvest and storage samples, respectively. The growth of Aspergillus flavus was 38-5-39-5% on the surface of the shells and 6–16% on the kernels without aflatoxin production. The contamination level of A flavus varied among samples collected from different regions. Peeling off the soft shell of pistachio nuts by hand reduced the contamination risk of A flavus to kernels. The predominant flora on stored pistachio nuts were Aspergillus, Penicillium, Cladosporium, Rhizopus, while the genera of Ulocladium, Trichothecium, Aureobasidium and Eurotium were less frequent. Thirty-five percent of the A flavus isolates produced aflatoxins on synthetic media.     

Effect of different ozone treatments on aflatoxin degradation and physicochemical properties of pistachios

This study evaluated the efficiency of ozone for the degradation of aflatoxins in pistachio kernels and ground pistachios. Pistachios were contaminated with known concentrations of aflatoxin (AF) B₁, B₂, G₁ and G₂. Pistachio samples were exposed to gaseous ozone in a chamber at 5.0, 7.0 and 9.0 mg L^?1 ozone concentrations for 140 and 420 min at 20 °C and 70% RH. Aflatoxin degradation was determined by high performance liquid chromatography (HPLC). The efficiency of ozone for aflatoxin degradation in pistachios increased with increasing exposure time and ozone concentration. The results indicated that AFB₁ and total aflatoxins could be reduced by 23 and 24%, respectively, when pistachio kernels were ozonated at 9.0 mg L^?1 ozone concentration for 420 min. Only a 5% reduction in AFB₁ and total aflatoxin levels could be achieved for ground pistachios under the same conditions. No significant changes occurred in pH, color, moisture content and free fatty acid values of pistachio kernels and ground pistachios. Fatty acid compositions of pistachios did not change significantly after the ozonation treatments. No significant changes were found between sweetness, rancidity, flavor, appearance and overall palatability of ozonated and non‐ozonated pistachio kernels. Significant changes were observed in the organoleptic properties of ground pistachios, except rancidity, after 5.0 mg L^?1 ozone treatment for 140 min. Ozonation was found to be more effective for degrading aflatoxins in pistachio kernels than ground pistachios. Copyright © 2006 Society of Chemical Industry     

Moisture-equilibrium relative humidity relationships in pistachio nuts with particular regard to control of aflatoxin formation

Adsorption and desorption isotherms have been determined both manometrically and by weight equilibration for Turkish pistachio nut kernel, shell and hull. Comparison of the calculated and experimentally determined adsorption isotherms for whole nuts showed good correlation. Nuts inoculated with Aspergillus flavus conidia were equilibrated to various ERH levels and stored in controlled environment cabinets at 28°C. Competitive growth of xerophilic strains of A. amstelodami prevented growth and aflatoxin production by the A. flavus at ERHs of 86% and below. At 88% ERH marked aflatoxin production occurred but competition was observed between the A. flavus and A. niger. In sealed containers metabolic moisture from growth of A. amstelodami raised the ERH from the initial 85% and permitted toxin production by A. flavus. The results are discussed in relation to post-harvest handling and storage of pistachio nuts.     

Consumers' ability to discriminate aflatoxin-contaminated Brazil nuts

The objectives of the study were to investigate the extent to which consumers can separate nuts with a high content of aflatoxin from sound nuts, and whether sorting results can be improved by information or whether they are affected by certain factors. A test panel consisting of 100 subjects was asked to crack 300 g Brazil nuts and to sort the nuts into those they considered edible and inedible. The test showed that consumers can, on current behaviour, discriminate aflatoxin-contaminated Brazil nuts to a significant extent. The median and the 95th percentile of the total concentrations of aflatoxins (B₁, B₂, G₁, G₂) in the samples before sorting were 1.4 and 557 µg kg^-1, respectively, and in the edible fractions after sorting 0.4 and 56 µg kg^-1, respectively. Given that levels of aflatoxins before sorting exceed either 2 µg aflatoxin B₁ kg^-1 or totally 4 µg aflatoxins kg^-1, there was no effect of aflatoxin concentrations before sorting on the probability of exceeding these thresholds in the edible fraction. This means that similar sorting results were obtained for samples with aflatoxin levels exceeding either of the two thresholds, irrespective of if the thresholds were exceeded with a few µg kg^-1 or up to more than 1000 µg kg^-1. None of the tested factors (such as sex, age, level of education, ethnic background or knowledge of mycotoxins) had any effects on the probability of exceeding either of the two aflatoxin thresholds.     

Spectral analyses of fluorescences in dent maize infected with Aspergillus flavus and Aspergillus parasiticus

Bright greenish yellow (BGYF) and blue white (BWF) fluorescences were associated with Aspergillus flavus and A. parasiticus infected maize. The fluorescences were studied spectrofluorometrically, the BGYF exhibiting a peak wave length between 480–485 nm and the BWF between 440–445 nm. Neither fluorescence varied in maize stored under different moistures and temperatures.

BWF was similar spectrally to the fluorescence of the endosperm of sound kernels but × 5 20 more intense. The spectrum of BWF was similar to Aflatoxin G₁ or a mixture of aflatoxin B₁, B₂, G₁ and G₂ when they were spotted on endosperm tissue. A color reference for BGYF was similar in peak wave length to BGYF. Amsoy soybeans without the seed coat fluoresced with a peak 470–475 nm and the intensity was low compared to BGYF in maize. A fluorescence of maize kernels visually similar to BGYF but not associated with Aspergillus infection or aflatoxin contamination was also investigated. This “false BGY” fluorescence was spectrally similar to the BGYF in infected kernels.     

Determination of aflatoxin risk components for in-shell Brazil nuts

A study was conducted on the risk from aflatoxins associated with the kernels and shells of Brazil nuts. Samples were collected from processing plants in Amazonia, Brazil. A total of 54 test samples (40 kg) were taken from 13 in-shell Brazil nut lots ready for market. Each in-shell sample was shelled and the kernels and shells were sorted in five fractions: good kernels, rotten kernels, good shells with kernel residue, good shells without kernel residue, and rotten shells, and analysed for aflatoxins. The kernel:shell ratio mass (w/w) was 50.2/49.8%. The Brazil nut shell was found to be contaminated with aflatoxin. Rotten nuts were found to be a high-risk fraction for aflatoxin in in-shell Brazil nut lots. Rotten nuts contributed only 4.2% of the sample mass (kg), but contributed 76.6% of the total aflatoxin mass (μg) in the in-shell test sample. The highest correlations were found between the aflatoxin concentration in in-shell Brazil nuts samples and the aflatoxin concentration in all defective fractions (R(2)=0.97). The aflatoxin mass of all defective fractions (R(2)=0.90) as well as that of the rotten nut (R(2)=0.88) were also strongly correlated with the aflatoxin concentration of the in-shell test samples. Process factors of 0.17, 0.16 and 0.24 were respectively calculated to estimate the aflatoxin concentration in the good kernels (edible) and good nuts by measuring the aflatoxin concentration in the in-shell test sample and in all kernels, respectively.     

Determination of aflatoxin risk components for in-shell Brazil nuts

A study was conducted on the risk from aflatoxins associated with the kernels and shells of Brazil nuts. Samples were collected from processing plants in Amazonia, Brazil. A total of 54 test samples (40?kg) were taken from 13 in-shell Brazil nut lots ready for market. Each in-shell sample was shelled and the kernels and shells were sorted in five fractions: good kernels, rotten kernels, good shells with kernel residue, good shells without kernel residue, and rotten shells, and analysed for aflatoxins. The kernel?:?shell ratio mass (w/w) was 50.2/49.8%. The Brazil nut shell was found to be contaminated with aflatoxin. Rotten nuts were found to be a high-risk fraction for aflatoxin in in-shell Brazil nut lots. Rotten nuts contributed only 4.2% of the sample mass (kg), but contributed 76.6% of the total aflatoxin mass (µg) in the in-shell test sample. The highest correlations were found between the aflatoxin concentration in in-shell Brazil nuts samples and the aflatoxin concentration in all defective fractions (R ^2?=?0.97). The aflatoxin mass of all defective fractions (R ^2?=?0.90) as well as that of the rotten nut (R ^2?=?0.88) were also strongly correlated with the aflatoxin concentration of the in-shell test samples. Process factors of 0.17, 0.16 and 0.24 were respectively calculated to estimate the aflatoxin concentration in the good kernels (edible) and good nuts by measuring the aflatoxin concentration in the in-shell test sample and in all kernels, respectively.     

Mycotoxins in edible tree nuts

Tree nuts (almonds, pistachios, and walnuts) are an exceptionally valuable crop, especially in California, with an aggregate value approaching $3.5 billion. Much of this economic value comes from overseas markets, with up to 60% of the crop being exported. The product can be contaminated with aflatoxins or ochratoxins, with the former being of special concern because of the strict regulatory levels (4 ppb total aflatoxins) applied by the European Community (EC). Natural, consumer-acceptable control methods are therefore required to conform to such limits. Research has shown that aflatoxin production is markedly decreased by the presence of natural antioxidants that occur in tree nuts, including hydrolysable tannins, flavonoids and phenolic acids. In vitro testing of individual compounds showed that the antiaflatoxigenic effect correlated with the structure and concentration of such compounds in individual nut varieties and species. This lead to the hypothesis that aflatoxin biosynthesis is stimulated by oxidative stress on the fungus and that compounds capable of relieving oxidative stress should therefore suppress or eliminate aflatoxin biosynthesis. Oxidative stress induced in A. flavus by addition of tert-butyl hydroperoxide to the media stimulated peak aflatoxin production and maintained high levels over time. However, aflatoxin formation was significantly inhibited by incorporation into the media of the antioxidant, tannic acid. Measures to increase natural products with antioxidant properties in tree nuts may thereby reduce or eliminate the ability of A. flavus to biosynthesize aflatoxins, thus ensuring levels at or below regulatory limits and maintaining export markets for U.S. tree nuts.     

Non-invasive detection of aflatoxin-contaminated figs using fluorescence and multispectral imaging

Agricultural products are prone to aflatoxin (AF)-producing moulds (Aspergillus flavus, A. parasiticus) during harvesting, drying, processing and also storage. AF is a mycotoxin that may cause liver cancer when consumed in amounts higher than allowed limits. Figs, like other agricultural products, are mostly affected by AF-producing moulds and these moulds usually produce kojic acid together with AF. Kojic acid is a fluorescent compound and exhibiting bright greenish yellow fluorescence (BGYF) under ultraviolet (UV) light. Using this fluorescence property, fig-processing plants manually select and remove the BGYF+ figs to reduce the AF level of the processed figs. Although manual selection is based on subjective criteria and strongly depends on the expertise level of the workers, it is known as the most effective way of removing AF-contaminated samples. However, during manual selection, workers are exposed to UV radiation and this brings skin health problems. In this study, we individually investigated the figs to measure their fluorescence level, surface mould concentration and AF levels and noted a strong correlation between mould concentration and BGYF and AF, and BGYF and surface. In addition to a pairwise correlation, we proposed a machine-vision and machine-learning approach to detect the AF-contaminated figs using their multispectral images under UV light. The figs were classified in two different approaches considering their surface mould and AF level with error rates of 9.38% and 11.98%, respectively.     

Evaluation of aflatoxin decontaminating by two strains of Saccharomyces cerevisiae and Lactobacillus rhamnosus strain GG in pistachio nuts

In this study, the surface binding ability of Saccharomayces cerevisiae and Lactobacillus rhamnosus GG (LBGG) to aflatoxin in pistachio nuts was compared. Results showed that Saccharomayces cerevisiae and Lactobacillus rhamnosus strains had aflatoxin binding ability of 40% and 35% with initial concentration of 10 ppb and 70% and 60% with initial concentration of 20 ppb aflatoxin, respectively. Acid treatment increased this ability for yeast and bacterium to 60% and 85% in first concentration and 73% and 90% for second concentration of aflatoxin, respectively. Also, heat treatment could raise surface binding of yeast to 55% and 75% for two concentrations. In addition, heat condition for Lactobacillus improved binding to 85% and 90% for two concentrations of aflatoxin. Experiments showed that microorganism’s immobilisation on contaminated pistachio had no effect on qualitative characteristics of pistachio such as colour, texture and peroxide value.     

Aflatoxins in nuts assayed by immunological methods

 Different kinds of raw and processed nuts available in the local retail market were investigated for aflatoxin content. Total aflatoxins and aflatoxin B₁ content were determined by immunoaffinity chromatography with fluorescence detection after reaction with bromine solution and by immunoenzymatic test kits. Of 29 investigated samples, 38% were contaminated. The total aflatoxin content in contaminated samples was between 1.20 μg/kg for peanuts and 5.50 μg/kg for walnuts. The concentration of aflatoxin B₁ found in contaminated samples was between 0.35 μg/kg for cashew nuts and 4.04 μg/kg for walnuts. The mean recovery of total aflatoxins was 95% for the Ridascreen test and 92% for immunoaffinity chromatography with fluorescence detection. For aflatoxin B₁ the mean recovery was 84%. Received: 4 March 1999 / Revised version: 17 May 1999     

Predicting the growth/no-growth boundary and ochratoxin A production by Aspergillus carbonarius in pistachio nuts

Black aspergilli are the main fungal contaminants in pistachio nuts. Ochratoxin A (OTA) production has been repeatedly reported in Aspergillus section Nigri; OTA has been occasionally detected in pistachio nuts in high concentrations. The aim of this study was to develop suitable validated models to predict the growth and OTA production boundaries by an Aspergillus carbonarius isolated from pistachios as a function of moisture content and storage temperature of pistachios. A full factorial design was used: the moisture content levels assayed were 12.5%, 17.9%, 24.0%, 29.5% and 34.8% and the incubation temperatures were 10, 15, 20, 25, 30, 37 and 42 degrees C. A probability model was built to predict the growth of the strain under the assayed conditions, which proved to be concordant in 73-91% of the cases with the observed probabilities in the validation test specifically chosen for limiting growth conditions. Similarly, the probability for the presence of OTA was correctly predicted in 90% of the cases. OTA accumulation was mainly a function of the temperature of storage, with a sharp increase at <15-20 degrees C; this value was very different from 30 to 35 degrees C, which was optimum for growth. Increasing OTA levels were found with increasing moisture content in the pistachio nuts. The impact of these results in the implementation of HACCP plans in Western Asian countries, is discussed. Probability models were applied in this work to mycotoxin accumulation for the first time, however, further research is required in the kinetics of mycotoxins accumulation to develop proper empirical models.     

Occurrence of Aflatoxins in Ground Red Chili Pepper and Pistachio Nut

In total, 240 samples were randomly collected from different locations in Gaziantep Turkey, 120 of which were unpacked and packed ground red chili pepper and 120 of which were unpacked and packed pistachio nuts. All samples of the unpacked and packed ground red chili pepper were found to be contaminated with aflatoxins and aflatoxin B1. Three (9.4%) of the packed ground red chili pepper samples were over the legal limit of aflatoxin B1 while 55 (62.5%) of the unpacked samples were over the legal limit. Thirty four (36.2%) of the unpacked pistachio nut samples were contaminated with aflatoxins. There is a need to strictly prohibit the use of batches of ground red chili pepper and pistachio nuts containing aflatoxins.     

Manual sorting to eliminate aflatoxin from peanuts

A manual sorting procedure was developed to eliminate aflatoxin contamination from peanuts. The efficiency of the sorting process in eliminating aflatoxin-contaminated kernels from lots of raw peanuts was verified. The blanching of 20 kg of peanuts at 140 degrees C for 25 min in preheated roasters facilitated the manual sorting of aflatoxin-contaminated kernels after deskinning. The manual sorting of raw materials with initially high aflatoxin contents (300 ppb) resulted in aflatoxin-free peanuts (i.e., peanuts in which no aflatoxin was detected). Verification procedures showed that the sorted sound peanuts contained no aflatoxin or contained low levels (<15 ppb) of aflatoxin. The results obtained confirmed that the sorting process was effective in separating contaminated peanuts whether or nor contamination was extensive. At the commercial level, when roasters were not preheated, the dry blanching of 50 kg of peanuts for 45 to 55 min facilitated the proper deskinning and subsequent manual sorting of aflatoxin-contaminated peanut kernels from sound kernels.     

Separating in-shell pistachio nuts from kernels using impact vibration analysis

A sorting system has been developed for the separation of small in-shell pistachio nuts from kernels without shells on the basis of vibrations generated when moving samples strike a steel plate. Impacts between the steel plate and the hard shells, as measured using an accelerometer attached to the bottom of the plate, produce higher frequency signals than impacts between the plate and the kernels. Signal amplitudes, on the other hand, were highly variable and by themselves were not useful for the separation of samples. An algorithm was developed using both amplitude and frequency information to classify the signals. The algorithm activated an air nozzle to divert in-shell nuts away from the kernel stream. A prototype sorter was tested at throughput rates of 0.33, 10, 20, and 40 nuts per second using a mix of 10% in-shell and 90% kernels. At the lowest throughput rate, classification accuracies were 96% for in-shell nuts and 99% for kernels. For throughput rates between 10 and 40 nuts/s, correct classification ranged from 84 to 90% for in-shell nuts. For kernels, accuracy was 95% at 10 and 20 nuts/s and 89% at 40 nuts/s.     

Determination of aflatoxin levels in nuts and their products consumed in South Korea

A total of 85 nuts and their products marketed in South Korea were assessed for aflatoxins using a monitoring scheme consisting of enzyme-linked immunosorbent assay (ELISA) for rapid screening, high performance liquid chromatography (HPLC) for quantification and LC–mass spectrometry (MS) for confirmation. Thirty-one out of 85 samples gave ELISA readings above 0.06 and were screened as possible positive samples. Aflatoxin contents of possible positive samples were determined using HPLC with a detection limit of 0.08–1.25 μg/kg and a quantification limit of 0.15–2.50 μg/kg. Nine samples including 1 raw peanut, 4 roasted peanuts, 2 peanut butters, 1 pistachio and 1 seasoned assorted nut were contaminated with aflatoxins (10.6% of incidence), ranging in various levels up to 28.2 μg/kg. LC–MS analysis on contaminated samples revealed that peaks eluting at 4.4, 5.2, 9.1 and 11.9 min were confirmed as aflatoxin G₁, aflatoxin B₁, aflatoxin G₂ and aflatoxin B₂, respectively.     

基于颜色特征的含黄曲霉毒素玉米颗粒的检出方法

为快速准确识别出感染黄曲霉毒素的玉米颗粒,基于黄曲霉毒素在365 nm紫外光照射下可发出黄绿色荧光(bright greenish-yellow fluorescence,BGYF)的特性,提出了一种以颜色为特征量的感染毒素颗粒的检出方法。首先针对同一组玉米颗粒样本分别获取其在紫外光和可见光下的2幅图像,对可见光图像进行二值化、颗粒区域填充等预处理操作,得到玉米颗粒的轮廓与位置信息;然后将进行彩色增强后的紫外光图像与预处理后的可见光图像掩膜;最后在掩膜图像中通过RGB模型中G通道下的阈值作为分割参数识别玉米颗粒上的感染区域。结果表明,所提出的识别方法对能够发出荧光的含黄曲霉菌玉米颗粒识别准确率达到84%以上,对含有黄曲霉毒素的正确检出率可达77.8%以上,能够达到区分检测的要求。