Effect of high hydrostatic pressure on Cryptosporidium parvum infectivity

The incidence of foodborne disease outbreaks caused by contaminated low-pH fruit juices is increasing. With recent mandatory pasteurization of apple juice and the industry's concerns of food safety, fruit juice processors are showing more interest in alternative nonthermal technologies that can kill >99.99% of microbial pathogens present in foods. The association of the coccidian protozoan, Cryptosporidium, with diarrheal disease outbreaks from contaminated tap water and fruit juice raises a safety concern in the food and beverage industries. The objective of this study was to evaluate the effects of high hydrostatic pressure (HHP) on C. parvum oocysts. Oocysts were suspended in apple and orange juice and HHP treated at 5.5 x 10(8) Pa (80,000 psi) for 0, 30, 45, 60, 90, and 120 s. Oocyst viability was assessed by excystation using bile salts and trypsin while the cell culture foci detection method was used to assess infectivity. Results indicated that HHP inactivated C. parvum oocysts by at least 3.4 log10 after 30 s of treatment. No infectivity was detected in samples exposed to > or =60 s of HHP and >99.995% inactivation was observed. This study demonstrated that HHP efficiently rendered the oocysts nonviable and noninfectious after treatment at 5.5 x 10(8) Pa.     

Inactivation of Cryptosporidium parvum oocysts in cider by flash pasteurization

Cryptosporidium parvum is a well-recognized pathogen of significant medical importance, and cider (apple juice) has been associated with foodborne cryptosporidiosis. This study investigated the effect of flash pasteurization on the viability of contaminant C. parvum oocysts. Cider inoculated with oocysts was heated at 70 or 71.7 degrees C for 5, 10, or 20 s, and oocyst viability was measured by a semiquantitative in vitro infectivity assay. By infecting multiple wells of confluent Madin-Darby bovine kidney cells with serial dilutions of heat-treated oocysts and examining infected cells by indirect fluorescent antibody staining, the most probable number technique was applied to quantify log reduction of oocyst viability. Heating for 10 or 20 s at either temperature caused oocyst killing of at least 4.9 log (or 99.999%), whereas oocyst inactivation after pasteurization for 5 s at 70 and 71.7 degrees C was 3.0 log (99.9%) and 4.8 log (99.998%), respectively. Our results suggested that current practices of flash pasteurization in the juice industry are sufficient in inactivating contaminant oocysts.     

Effect of organic acids and hydrogen peroxide on Cryptosporidium parvum viability in fruit juices

Cryptosporidium parvum has historically been associated with waterborne outbreaks of diarrheal illness. Foodborne cryptosporidiosis has been associated with unpasteurized apple cider. Infectious oocysts are shed in the feces of common ruminants like cattle and deer in and near orchards. In this study, the ability of organic acids and hydrogen peroxide (H2O2) added to fruit juice to inhibit the survival of C. parvum was analyzed. Oocyst viability was analyzed by a cell culture infectivity assay with the use of a human ileocecal cell line (HCT-8) whose infectivity pattern is similar to that for human oral infectivity. Cell monolayers were infected with 10(6) treated oocysts or a series of 10-fold dilutions. Parasitic life stages were visualized through immunohistochemistry with 100 microscope fields per monolayer being counted. In vitro excystation assays were also used to evaluate these treatments. Organic acids and H2O2 were added to apple cider, orange juice, and grape juices on a weight/volume basis. Malic, citric, and tartaric acids at concentrations of 1 to 5% inhibited C. parvum's infectivity of HCT-8 cells by up to 88%. Concentrations ranging from 0.025 to 3% H2O2 were evaluated. The addition of 0.025% H2O2 to each juice resulted in a >5-log reduction of C. parvum infectivity as determined with a most-probable-number-based cell culture infectivity assay. As observed with differential interference contrast and scanning electron microscopy, reduced infectivity may be mediated through effects on the oocyst wall that are caused by the action of H2O2 or related oxygen radicals. The addition of low concentrations of H2O2 can represent a valuable alternative to pasteurization.     

Detection of Cryptosporidium parvum oocysts on fresh vegetables and 1 herbs using antibodies specific for a Cryptosporidium parvum viral antigen

The purpose of this study was to determine if the viral symbiont of Cryptosporidium parvum (CPV) sporozoites could be used as a target for sensitive detection of the parasite in food samples. Polyclonal sera specific to the recombinant viral capsid protein (rCPV40) was used in a dot blot hybridization assay to detect oocysts recovered from green onions and cilantro. Small batches of chopped green onions and cilantro leaves were artificially contaminated with three different concentrations of oocysts: 10(6), 10(2), and 10(1). rCPV40 was superior in detecting oocysts compared with other antibodies directed toward total oocyst protein and oocyst surface antigens. This study provides evidence that CPV is an excellent target for sensitive detection of C. parvum oocysts in foods.     

Simultaneous detection of Escherichia coli O157:H7, Salmonella, and Shigella in apple cider and produce by a multiplex PCR

With three pairs of primers, a multiplex PCR assay was established for the simultaneous detection of Escherichia coli 0157:H7, Salmonella, and Shigella. Under the optimized conditions, the assay yielded a 252-bp product from E. coli O157:H7, a 429-bp product from Salmonella Typhimurium, and a 620-bp product from Shigella flexneri, respectively. When the DNA extraction of multiple target organisms was included in the same reaction, two or three corresponding amplicons of different sizes were observed. In the specificity test, 10 E. coli O157:H7 strains and one E. coli O157:NM strain showed the expected 252-bp amplicon. Seven other E. coli strains yielded no signal. Additionally, the 429-bp amplicon was produced from 20 Salmonella strains covering 16 serotypes, whereas the 620-bp amplicon was generated from 11 Shigella strains covering 4 species. No nonspecific amplification was observed with DNA from 48 other bacterial strains. Following a 24-h enrichment, the developed assay could concurrently detect the three pathogens at initial inoculation levels of approximately 8 x 10(-1) CFU/g (or CFU/ml) in apple cider, cantaloupe, lettuce, tomato, and watermelon and 8 x 10(1) CFU/g in alfalfa sprouts. The whole procedure can be easily completed within 30 h. The multiplex PCR assay can potentially be a simple, rapid, and efficient tool for presumptive and simultaneous screening of apple cider and produce for contamination by E. coli O157:H7, Salmonella, and/or Shigella.     

Optimization of DNA extraction and molecular detection of Cryptosporidium oocysts in natural mineral water sources

The numerous published methods for extracting DNA from Cryptosporidium oocysts for PCR identify the lack of an optimized standard method for clinical, environmental, and public health investigations of cryptosporidiosis. A method that maximizes DNA extraction reliably, particularly from small numbers of partially purified or purified oocysts present in mineral waters and environmental samples, is required. We describe a maximized method for liberating DNA from Cryptosporidium parvum oocysts by 15 cycles of freezing (liquid nitrogen) and thawing (65 degrees C) in lysis buffer containing sodium dodecyl sulfate. The inhibitory effects of sodium dodecyl sulfate are abrogated by the addition of Tween 20 to the PCR reaction. We tested seven different C. parvum oocyst isolates, consistently detecting fewer than five oocysts following direct PCR amplification of a segment of the 18S rRNA gene. Older oocysts, which were more refractory to freeze-thawing, were disrupted effectively. A single oocyst in each of two mineral water concentrates was detected by both microscopy and PCR/Southern blotting. We recommend 15 cycles of freeze-thawing, with thawing at 65 degrees C in lysis buffer, to maximize oocyst disruption and DNA extraction, particularly when isolate history and oocyst age are unknown. Both the DNA extraction method and the PCR described can be used for clinical, environmental, and public health investigations of cryptosporidiosis.     

Direct detection of Cryptosporidium parvum oocysts by immunomagnetic separation-polymerase chain reaction in raw milk

Cryptosporidium parvum is an emerging protozoan parasite responsible for several serious outbreaks of cryptosporidiosis, an enteric infection characterized by severe intestinal distress. This parasite can be transmitted through contaminated water and raw food in the oocyst form, which is resistant to many environmental stresses and food processes. C. parvum is also commonly found on dairy farms and could be transmitted to humans through contaminated raw milk and dairy products. Thus, an immunomagnetic separation-polymerase chain reaction assay for direct detection of C. parvum oocysts in milk was developed. The procedure was able to detect < 10 C. parvum oocysts. Thus, it could be used for monitoring milk samples.     

Detection of fruit juice adulteration in apple and pear juice

A method has been developed to detect the adulteration of pear juice with apples and apple juice with pears. In different varieties of apples and pears and in several commercial juices the flavonoids were examined by HPLC with respect to their quality and quantity. The chalcones phloretin glucoside and phloretin xyloglucoside are typical compounds found in apples (detection limit 7 ng). They are suitable indicators for detecting adulteration of pear juice with apples. Isohamnetin glucoside cannot be detected in apples (detection limit 10 ng), but can be used to detect pears in apple juice. The extract or juice was purified with the aid of a polyamide column. The evaporated eluate of methanol was analysed by HPLC (gradient: acetonitrile/1% acetic acid). An RP-18 column and a UV-detector were used. Additionally, the UV spectra of the compounds indicating an adulteration were recorded by a diode array detector.     

Optimization of a fluorescence sandwich enzyme-linked immunosorbent assay for detection of Escherichia coli O157:H7 in apple juice

Sandwich enzyme-linked immunosorbent assay, especially when coupled with biosensor technology, is a simple methodology that can rapidly screen juices for Escherichia coli O157:H7 contamination. However, sampling directly from apple juice and ciders has been postulated to reduce immunoassay sensitivity. In fluorescence sandwich enzyme-linked immunosorbent assays using commercially available polyclonal or monoclonal antibodies, sampling pasteurized apple juice spiked with E. coli O157:H7 compared to spiked phosphate-buffered saline shifted the range of detection. The spiked apple juice range of detection was 10(4) to 10(6) CFU/ml, whereas that for spiked phosphate-buffered saline was 10(6) to 10(8) CFU/ml, representing a hundredfold difference in sensitivity. Apple juice also increased background fluorescence intensity (P < 0.001) while reducing the net fluorescence intensity per CFU (P < 0.001). The addition of the polymer polyvinylpyrrolidone to apple juice significantly improved assay performance by increasing sensitivity and net fluorescence intensity per CFU and by reducing background fluorescence. Adjusting pH of apple juice from 3.9 to 7.4 improved assay performance but not to the degree seen with phosphate-buffered saline or polyvinylpyrrolidone-treated apple juice samples. The apple juice polyphenol, epicatechin, reduced net fluorescence intensity in a concentration-dependent manner, a change that was reversed by polyvinylpyrrolidone. Taken all together, these results suggest that polyvinylpyrrolidone can improve detection of O157:H7 in juices by reducing the effect of polyphenols on fluorescence sandwich enzyme-linked immunosorbent assay performance.     

Haze Development in Aerobically or Anaerobically Produced Clarified Apple Juices

Haze production in aerobically or anaerobically produced Red Delicious apple juice was assessed following heating, aeration, and protein addition. Stored aerobic juice produced haze in 6 wk, but anaerobic juice was haze-free unless oxygen was introduced. Phenolic material was incorporated into haze in both systems. HPLC (detection at 420 nm) of concentrated juice and addition of 100 mg/L BSA suggested the presence in both juices of ‘reactive’ material which could complex and precipitate during storage. Phenolic compounds, heating, and especially oxidation during processing (and storage) appear to be major determinants of ‘haze potential’ in clarified apple juice.     

Detection of Cryptosporidium and Giardia in molluscan shellfish by multiplexed nested-PCR

A multiplexed nested-PCR procedure (ABC-PCR) previously developed to detect Cryptosporidium spp. and Giardia duodenalis assemblages A and B in whole human faeces was applied to DNA extracted from filter-feeding molluscs. Species of Cryptosporidium and G. duodenalis were identified by restriction fragment analysis of the PCR products and by DNA sequencing. The extraction and ABC-PCR procedures were shown to be suitable for application to shellfish by amplification of specific target sequences using DNA from Cryptosporidium parvum genotype 2 and G. duodenalis assemblages A and B which were spiked into DNA extracted from mussels. Using 49 molluscan shellfish specimens (18 clam, 22 mussel and 9 oyster samples) from Spain, cryptosporidial oocysts were detected in 56% by immunofluorescence microscopy, and in 44% by ABC-PCR. For detection of Cryptosporidium, there was a significant association, but not total agreement, between the results of microscopy and PCR. G. duodenalis assemblage B was detected from one oyster sample by PCR. Amongst 38 specimens (20 mussel and 18 cockle samples) collected in the UK and tested by the ABC-PCR, G. duodenalis was not detected, and Cryptosporidium was detected in 11% of the samples. Overall, the 26 samples where Cryptosporidium was detected, C. hominis/C. parvum genotype 1 was detected in 1, C. parvum genotype 2 in 22, and the remaining three samples contained either sequences similar to C. parvum genotype 2 or heterogeneous mixtures of Cryptosporidium species. There was no significant association between the level of Escherichia coli detected by conventional microbiological methods and the presence of Cryptosporidium detected by ABC-PCR.     

微波提取苹果汁中脂环酸芽孢杆菌DNA的PCR检测方法研究

根据Genbank公布的酸土脂环酸芽孢杆菌(Alicyclobacillus acidoterrestris DSM 3922T)鲨烯环化酶序列自行设计一对引物,利用微波技术直接从果汁样品中提取目标菌DNA,对引物特异性、微波法提取DNA的扩增效果及检测灵敏度进行探讨。结果表明：微波功率1000W、处理时间30s、经5000r/min离心2min即可获得酸土脂环酸芽孢杆菌基因组DNA,所得模板质量符合聚合酶链式反应检测要求,目的条带清晰,检测时间仅为2h,检测限为200CFU/mL,有望真正应用于苹果汁生产的在线检测。     

Survival of Escherichia coli O157:H7 and Salmonella in apple cider and orange juice as affected by ozone and treatment temperature

Inactivation of Escherichia coli O157:H7 and Salmonella in apple cider and orange juice treated with ozone was evaluated. A five-strain mixture of E. coli O157:H7 or a five-serovar mixture of Salmonella was inoculated (7 log CFU/ml) into apple cider and orange juice. Ozone (0.9 g/h) was pumped into juices maintained at 4 degrees C, ambient temperature (approximately 20 degrees C), and 50 degrees C for up to 240 min, depending on organism, juice, and treatment temperature. Samples were withdrawn, diluted in 0.1% peptone water, and surface plated onto recovery media. Recovery of E. coli O157:H7 was compared on tryptic soy agar (TSA), sorbitol MacConkey agar, hemorrhagic coli agar, and modified eosin methylene blue agar; recovery of Salmonella was compared on TSA, bismuth sulfite agar, and xylose lysine tergitol 4 (XLT4) agar. After treatment at 50 degrees C, E. coli O157:H7 populations were undetectable (limit of 1.0 log CFU/ml; a minimum 6.0-log CFU/ml reduction) after 45 min in apple cider and 75 min in orange juice. At 50 degrees C, Salmonella was reduced by 4.8 log CFU/ml (apple cider) and was undetectable in orange juice after 15 min. E. coli O157:H7 at 4 degrees C was reduced by 4.8 log CFU/ml in apple cider and by 5.4 log CFU/ml in orange juice. Salmonella was reduced by 4.5 log CFU/ml (apple cider) and 4.2 log CFU/ml (orange juice) at 4 degrees C. Treatment at ambient temperature resulted in population reductions of less than 5.0 log CFU/ml. Recovery of E. coli O157:H7 and Salmonella on selective media was substantially lower than recovery on TSA, indicating development of sublethal injury. Ozone treatment of apple cider and orange juice at 4 degrees C or in combination with mild heating (50 degrees C) may provide an alternative to thermal pasteurization for reduction of E. coli O157:H7 and Salmonella in apple cider and orange juice.     

Clarification of apple juice by electroflotation

Apple Juice industry is in search of a simplified technology which enables a quick clarification and stabilisation of apple juice. This study aimed to evaluate the potential of electroflotation as an alternative for the clarification of apple juice. Clarification of apple juice by electroflotation was first done at various current densities (10, 20 and 40 mA/cm²) with and without addition of gelatin (200 mg/l). Afterwards, the electroflotation treatments were done at a current density of 20 mA/cm² with various concentrations of added gelatin (0, 50, 100 and 200 mg/l). It was shown that electroflotation treatments alone was efficient to reduce the tannin and protein contents of apple juice. However, the decrease in the protein content was in large part due to the use of pectinases prior to the electroflotation treatments. The use of gelatin in combination with the electroflotation aided in the clarification process. The highest gelatin concentration used in this study (200 mg/l) resulted in a better reduction of tannin and protein levels, while a current density of 20 mA/cm² was found to be optimal. Turbidity observed in the juices clarified with electroflotation treatments was in average lower than 10 NTU but higher than 2 NTU which is generally required to produce a stable clarified juice. Brix degree and pH of the apple juice was not affected by the electroflotation treatments while the color was improved.

Industrial relevance

The production of clarified and stable apple juice is a subject of interest for the beverages industries. The clarification step which remained long and discontinuous implied the addition of a large quantity of pectolytic enzyme and of clarifying agents (such as gelatin) to the freshly pressed juice to induce the precipitation of proteins and other suspended matter in 15–20 h. Fining treatments were followed by a separation step usually consisting of decantation and classical filtration on filter-press, or flotation by dispersed gas. The development of membrane separation processes to replace the traditional approach has enabled the automation of the whole production resulting in lower labor requirement and a considerably shorter process time than the traditional process.However, the performance of membrane separation processes is influenced by the declining permeate flux with time, which is due to membrane fouling. In some instances, permeate flux decline makes membrane separation processes unattractive for the clarification of apple juice. To our knowledge, we are the first research group to use electroflotation (EF) for clarification of apple juice. It was shown that EF treatments alone were efficient to reduce the tannin and protein contents of apple juice. In addition, the use of gelatin in combination with the EF aided in the clarification process. Turbidity observed in the juices clarified with EF treatments for 30 min was in average lower than 10 NTU. Brix degree and pH of the apple juice were not affected by the EF treatments while the color was improved.When compared to the values reported in the literature for flotation by dispersed gas, it seems that EF shows better efficiency than flotation in decreasing the juice turbidity (99% decrease for EF as compared to 90% decrease for flotation). In addition, for experiments carried out by conventional flotation larger amount of fining agent are used (70–150 mg of gelatin/l, 400–800 mg/l of silica sol and 200–500 mg/l of bentonite). For these reasons, the new process we propose is advantageous when compared to the traditional flotation approach and it should have a measurable impact on the advancements in the production of clarified apple juice. If used as a pre-treatment to ultrafiltration clarification, it is expected that it would reduce membrane fouling resulting in higher productivity.     

Electrophoretic and immunochemical determination of bioindustrial enzymes in apple juice

The present study was undertaken to develop a specific and sensitive method which allows the detection of trace amounts of the potentially allergenic bioindustrial enzymes Pectinex Ultra-SPL (pectinase) and Gamylo 200L (amylase) in apple juice. This was achieved by an immunoblotting procedure based on sodium dodecyl sulfate (SDS) electrophoretic separation, followed by an immunochemical detection step using polyclonal rabbit antisera directed against these enzymes and, secondly, alkaline phosphatase labelled antibodies in combination with a highly sensitive enhanced chemiluminescent (ECL) detection method. In a juice spiked with enzyme at various concentrations between 10,000 and 1 ppm (1.0-0.0001%), the detection limit was 10 ppm (=0.001%) for Pectinex Ultra-SPL and 1 ppm (=0.0001%) for Gamylo 200L, respectively. Since the enzyme preparations contain less than 5% of enzymes, the detection limit for the pure enzymes is markedly lower, at least below 1 ppm. Whereas in the samples withdrawn during the initial stages of the industrial production process of apple juice, Pectinex Ultra-SPL and Gamylo 200L components were detectable in concentrations between 50 and 100 ppm, after clearing, ultrafiltration and pasteurization, neither pectinase nor amylase was detectable in the final consumer's product. Under the conditions employed, the bioindustrial enzymes Pectinex Ultra-SPL and Gamylo 200L were virtually removed during the course of production. The risk of sensitizing consumers by intake of apple juice seems to be marginal. This view is supported by the results of an animal model (anaphylaxis test), in which neither gastro-enteral reactions in guinea pigs and mice, nor IgE or IgG production by orally feeding these animals with apple juice concentrate, could be induced.     

Best Estimated Aroma and Taste Detection Threshold for Guaiacol in Water and Apple Juice

ABSTRACT: Alicyclobacillus acidoterrestris can produce sufficient guaiacol (methoxyphenol), a metabolic by-product of the bacterium, in apple juice to cause a detectable taint characterized by an antiseptic off-odor or distinct medicinal flavor and lingering aftertaste. Bacterial spoilage may not be visibly detectable. The objective of this study was to determine the best estimate threshold (BET) for detection of guaiacol in water and commercial pasteurized apple juice from concentrate using the forced-choice ascending concentration method of limits with an experienced 17-member sensory panel. The mean BET for aroma detection of guaiacol in water and apple juice was 0.48 ppb and 0.91 ppb, respectively. The mean BET for taste detection of guaiacol in water and apple juice was 0.17 ppb and 0.24 ppb, respectively. Individual aroma BET values ranged from 0.06 ppb to 4.71 ppb guaiacol in water and 0.17 ppb to 4.71 ppb for guaiacol in apple juice. Individual taste BET values ranged from 0.01 ppb to 4.71 ppb for guaiacol in water and apple juice. The taste BET was equal to or lower than the aroma BET for guaiacol in both water and apple juice for all panelists. There was about a 500-fold range in guaiacol taste detection between panelists, with some individuals exhibiting a BET value as low as 10 ppt (trillion). The information should be useful for developing quality assurance sensory methodology to evaluate potential apple juice flavor spoilage by Alicyclobacillus spp.     

Towards standard methods for the detection of Cryptosporidium parvum on lettuce and raspberries. Part 1: development and optimization of methods

No standard method is available for detecting protozoan parasites on foods such as soft fruit and salad vegetables. We report on optimizing methods for detecting Cryptosporidium parvum on lettuce and raspberries. These methods are based on four basic stages: extraction of oocysts from the foodstuffs, concentration of the extract and separation of the oocysts from food materials, staining of the oocysts to allow their visualization, and identification of oocysts by microscopy. The concentration and separation steps are performed by centrifugation, followed by immunomagnetic separation using proprietary kits. Oocyst staining is also performed using proprietary reagents. The performance parameters of the extraction steps were extensively optimized, using artificially contaminated samples. The fully developed methods were tested several times to determine their reliability. The method to detect C. parvum on lettuce recovered 59.0+/-12.0% (n=30) of artificially contaminated oocysts. The method to detect C. parvum on raspberries recovered 41.0+/-13.0% (n=30) of artificially contaminated oocysts.     

Infectivity of Cryptosporidium parvum oocysts after storage of experimentally contaminated apples

Irrigation water and washing water have been inferred to be associated with contamination of fresh fruits and vegetables with pathogenic microorganisms infectious for humans. The objective of the present study was to determine whether apples experimentally contaminated with Cryptosporidium oocysts represent a food safety concern. Laser scanning confocal microscopy revealed no morphological changes in Cryptosporidium parvum oocysts attached to apples after 6 weeks of cold storage, suggesting that oocysts might remain viable and possibly infectious during prolonged storage. Mice were fed apple peels from experimentally contaminated apples to determine whether oocysts had remained infectious on apples stored for 4 weeks. All mice developed cryptosporidiosis. To evaluate the strength of oocyst attachment to apples, washing methods that have been reported to be helpful for recovery of oocysts from various foodstuffs were evaluated, except that the intensity of washing was increased in the present study. None of the tested washing methods succeeded in completely removing oocysts from the apple peel. The most efficient removal (37.5%) was achieved by rigorous manual washing in water with a detergent and by agitation in an orbital shaker with Tris-sodium dodecyl sulfate buffer. Glycine and phosphate-buffered saline buffers had no effect on oocyst removal. Scanning electron microscopy revealed that some oocysts were attached in deep natural crevices in the apple exocarp and others were attached to the smooth surface of the peel. Some oocysts were closely associated with what appeared to be an amorphous substance with which they might have been attached to the apple surface.     

N-methyl carbamate concentrations and dietary intake estimates for apple and grape juices available on the retail market in Canada.

Infants and young children consume fruit juices and drinks at rates exceeding those of older children and adults. Carbamate pesticides are known to be used on a broad spectrum of crops, including orchard and vine crops such as apples and grapes. Concern over potential exposure to these acutely toxic pesticides by infants and young children has increased in the last decade. Liquid chromatography with fluorescence detection was used to determine the concentrations of seven N-methyl carbamates and three transformation products in domestic and imported apple and grape juices collected across Canada. Carbaryl was the most frequently (58.6%) detected N-methyl carbamate in juice samples studied. It was observed more frequently in grape juices than in apple or mixed juices. Oxamyl and methomyl were detected in apple juice samples, although they were below detection limits in all grape and mixed juice samples analysed. Maximum levels of carbaryl, methomyl and oxamyl were 93, 6.7 and 4.6 ng ml(-1), respectively. All other analytes were not present in any juice sample at concentrations above the method detection limit (0.3 ng ml(-1)). In all cases, N-methyl carbamate residues were well below the maximum residue limit established for apples and grapes in the Canadian Food and Drug Regulations. No estimated dietary intakes were above the acceptable daily intakes in any age-sex category, where an acceptable daily intake has been proposed. Carbaryl short-term intake estimates were calculated and all were below the proposed acute reference doses.     

Quantitative analysis of patulin in apple juice by thin-layer chromatography using a charge coupled device detector

A method was developed and validated in-house for the detection and quantification of patulin in apple juice concentrate using a charge coupled device (CCD) on thin-layer chromatography (TLC) plates. Samples were extracted with ethyl acetate and then cleaned-up by extraction with a sodium carbonate solution. The method showed a mean recovery of 95%. The quantification and detection limit were 14 µg l^?1 and 0.005 µg per spot, respectively. The CCD camera is sufficiently sensitive to detect changes in spot fluorescence intensity caused by small differences in mycotoxin concentration under homogeneous illumination from a UV light source. The results of validation confirmed the efficiency of the method, which is sensitive enough to be used to quantify patulin in apple juice by producers or for government monitoring/survey programs. The method was applied to the analysis of 16 apple juice concentrate samples and patulin levels ranged from 15 to 46 µg l^?1. This study demonstrated the applicability of the TLC–CCD technique as a tool for monitoring patulin in apple juice.