Effect of processing stages of apple juice concentrate on patulin levels

The effects of different stages of apple juice concentrate production on patulin levels were investigated. Patulin was detected in all samples analyzed in concentrations ranging from 56 to 653 μg/L. Apple paste resulted from milling process had high levels of patulin. The results of this study indicate that it is possible to reduce patulin level in apple juices. After pasteurization, enzymatic treatment, microfiltration and evaporation processes, the mean loss of patulin was 39.6, 28.3, 20.1 and 28.4%, respectively. When apple juices concentrate were diluted from 69 to 12°Brix to consume, patulin content ranged from 15 to 46 μg/L. Patulin content in all juice samples was lower than the limit of 50 μg/L considered acceptable by the Codex Alimentarius Commission. But if consider the maximum permitted concentration established for apple products intended for infants and young children by The Commission of the European Communities all samples were found to exceed patulin concentration of 10 μg/L.     

Molecularly imprinted polymer for extraction of patulin in apple juice samples

A new molecularly imprinted polymer (MIP) was prepared in a two-step process. First, SiO₂-γ-MPTS was obtained by mixing γ-methacryloxypropyltrimethoxysilane (γ-MPTS) and tetraethoxysilane (TEOS). Second, SiO₂-γ-MPTS was polymerized in the presence of patulin (PAT) as a template, maleic acid (MA) as a functional monomer, ethylene glycol dimethacrylate (EGDMA) as a cross linker, 2,2-azobis- (2- methylpropionitrile) (AIBN) as a precursor and acetonitrile as a porogen solvent. The prepared sorbent was successfully applied to selective solid phase extraction (SPE) of PAT in containing apple juices and the MIP@SPE method was validated. The optimum conditions for PAT extraction using the novel MIP@SPE were: 50 mg mass of adsorbent, sodium bicarbonate with (1%) acetic acid as washing solvent and 5 mL acetonitrile as eluting solvent. The developed MIP@SPE method had high selectivity and good affinity toward PAT and the recoveries of the target analyte were in the range of 82–98% with a precision of 3.03%–3.83% (RSD). In addition, a good linearity (r² > 0.99) within the range 0.1–10 mg L⁻¹ and a low LOD (8.6 μg L⁻¹) and LOQ (28.6 μg L⁻¹) were obtained. The test of reusability showed good results for at least 6 cycles.     

Single-laboratory validation of a liquid chromatography method for the determination of patulin in apple juice

A method for patulin determination in apple juice was optimized and single-laboratory validated. Patulin was extracted with ethyl acetate and the extracts were cleaned-up in a column prepared in the laboratory. The determination was carried out by liquid chromatograph with a C₁₈ column and diode array detector. Linearity was observed from 25.4 to 532.5 μg/L. Mean recovery of 81.6% and relative standard deviations <5.7% were obtained for samples spiked with 27, 58 and 122 μg/L. Limits of quantification and detection were 10 and 4 μg/L, respectively. In the ruggedness experiment, seven factors were considered. The method presented the advantages of easiness, economical aspects, patulin identity confirmation and use of non-toxic solvents and reagents.     

Incidence of patulin contamination in apple juice produced in Iran

Patulin is a secondary metabolite produced mainly in rotten parts of apples by a wide range of fungi. Due to its mutagenic and teratogenic nature and possible health risks to consumers, many countries have regulations to reduce level of patulin in apple products to as low as practically possible. A survey of the presence of patulin was conducted in 2002 on 65 locally produced commercial apple juice and apple juice concentrates purchased from retail outlets or producers in Iran. In this study 42 samples of apple juice and 23 apple juice concentrate surveyed for patulin contamination. Results of the study showed in 69% of apple juice and 78% of the apple juice concentrates level of patulin found was higher than 15 μg/l. Overall 33% of the apple juice samples had patulin levels higher than 50 μg/l with maximum level of 285 μg/l and 56% of the apple juice concentrates with patulin level of higher than 50 μg/l and maximum level of 148 μg/l. Although the mean concentration of patulin in apple juice samples surveyed was slightly lower than maximum level recommended by the Codex Alimentarious, fairly high incidence of patulin in these samples indicates the need for improving production techniques by industry in order to reduce the incidence and level of patulin contamination in apple juices.     

Solid-phase extraction and HPLC determination of Ochratoxin A in cereals products on Chilean market

Ochratoxin A (OTA) is a mycotoxin produced by different species of Aspergillus and Penicillium fungi. The presence of this mycotoxin in cereals-based products has relation with manufacturing practices, especially with storage conditions. An extraction procedure for OTA from wheat-based products was implemented in this study. The method uses an alkaline extraction with NaHCO₃, purification with Sep-Pak^® RP-18 cartridges; and quantitative analysis by high performance liquid chromatography with fluorescence detection. The presence of OTA was confirmed by the formation of Ochratoxin A methyl ester. The method shows good validation parameters with a rate of recovery rate over 95%, limits of detection and quantification of 0.6 and 2.1 μg kg^?1, respectively. Once the method was validated; 31 samples including, flour, corn starches and rice were analyzed. About 70% of flour samples, 50% of rice and 63% of corn starch samples resulted positives for OTA.     

Effective biosorption of patulin from apple juice by cross-linked xanthated chitosan resin

In this work, cross-linked xanthated chitosan resin (CXCR) was prepared by inverse suspension cross-linking method, using glutaraldehyde as cross-linking agent and carbon disulfide as modification agent. The biosorption of CXCR for patulin from apple juice was investigated. Batch adsorption experiments were performed to evaluate the effect of pH, temperature, contact time and initial concentration. The adsorption of CXCR for patulin was characterized by Fourier Transform Infrared Spectroscopy (FTIR). The results showed that the optimum adsorption conditions of CXCR for patulin was achieved at pH 4, 30 °C for 18 h. The adsorption data could be fitted with pseudo–second order kinetic model and Freundlich isotherm model. It indicated that CXCR is an appropriate adsorbent for the removal of patulin from apple juice.     

Survey of patulin in apple juice concentrates in Shaanxi (China) and its dietary intake

This study surveyed the patulin level in the apple juice concentrates from Shaanxi province for four consecutive processing seasons, and evaluated the dietary exposure to this mycotoxin through apple juice. A total of 1987 apple juice concentrate samples were analyzed by high-performance liquid chromatography with a diode-array detector, and only 4 samples in 2007–2008 processing season contained patulin above 50 μg/kg, the current regulatory limit in apple juice. The incidence of patulin contamination varied significantly from 96.7% to 100% (P < 0.05) by processing season, and the patulin level among the four processing seasons varied significantly with the median level from 6.30 to 8.90 μg/kg (P < 0.05, Kruskal–Wallis test). The highest evaluated patulin intake based on the apple juice consumption from the World Health Organization's Global Environment Monitoring System/Food Contamination Monitoring and Assessment Programme (GEMS/Food) was 1.04 ng/kg body weight (bw)/day in cluster diet F. And the patulin intakes for apple juice consumers among adults, children and babies were estimated to be 28.1, 67.5 and 110 ng/kg bw/day. All of the above values were well below the provisional maximum tolerable daily intake proposed by the Joint FAO/WHO Expert Committee on Food Additives.     

Double antibodies sandwich enzyme-linked immunosorbent assay for the detection of Alicyclobacillus acidoterrestris in apple juice concentrate

Two heterologous antibodies against Alicyclobacillus acidoterrestris (A. acidoterrestris) were obtained from Sprague Dawley (SD) Rats and Japan White Rabbits by immunizing them with an A. acidoterrestris strain isolated from apple juice concentrate (AJC). By using these two antibodies, we established a double antibodies sandwich ELISA (DAS-ELISA) method for the rapid detection of A. acidoterrestris in AJC, with the detection limit of 5 × 10³ CFU/mL. There was no cross-reaction with seven common foodborne microorganisms by employing this established essay, and the coefficient of variation (CV) within and among independent runs were less than 5.0% and 10% respectively, indicating of satisfactory specificity and repeatability. This DAS-ELISA assay was successfully applied to detect A. acidoterrestris in AJC samples when coupled with selective incubation for 12 h, and the results were agreed well with those obtained from the routine K medium method. Further, this method shows advantage over indirect ELISA (ID-ELISA) and SPA-ELISA assays previously reported by our group, facilitating the use of this newly developed assay method in food products.     

Occurrence of patulin in various fruit juice marketed in Tunisia

Patulin (PAT) is a secondary metabolite, which is mainly produced by certain species of Aspergillus and Penicillium fungi. The objective of this study is to investigate the presence of Patulin in various fruit products marketed and consumed in Tunisia. A total of 214 samples (30 concentrated juice samples, 42 apple juice samples, 42 pear juice samples, 34 mixed juice samples, 35 compote samples, 15 apple jams samples and 16 pear jam samples) were analyzed. The incidence of PAT contamination was 50% with a concentration ranging from 2 to 889 μg/l with an average of 89 μg/l and a median of 41 μg/l. Twenty two percent (22%) of total analyzed samples exceeded the limit recommended by the European Union. Our results showed that PAT seems to be a problem in fruit products marketed in Tunisia, which required and recommended a hard and fast surveillance of this toxin.     

Detection of addition of apple juice in purple grape juice

Due to increasing grape juice consumption, quality control is a reality in all countries which produce and consume this product. The most common form of adulteration is by substituting grape juice with apple juice. The adulterated samples can be identified by specific analysis, since apple juice has some compounds that grape has not. There are many studies about the phlorizin and sorbitol content in wines, but in grape juice they are scarce. Therefore, analytical methods to identify the composition of grape juices and determine their authenticity are needed to ensure quality control. The present study aimed to the validation and application of analytical methods for the determination of phlorizin and sorbitol, to detect the addition of apple juice in purple grape juice, by high-performance liquid chromatography. Initially, the methods were validated and tests were conducted by additions of percentages of apple juices in grape juices. Finally, experimental and commercial grape and apple juices were analyzed. Our study found that both methods are effective in detecting the addition of apple juice in grape juice. Four of the 39 commercial grape juices analyzed showed adulteration by the addition of apple juice in grape juice.     

Effect of processing of apple puree on patulin content

Mycotoxin patulin represents potential health hazard especially in apple based baby food. There are already a lot of data to predict the fate of the toxin during the processing of fruits, but not enough for the apple puree and the existing data for apple puree are not usually consistent. The objective of the study was to evaluate the course of patulin degradation during the processing of apple puree under the conditions close to the real production process. The apple samples were spiked with patulin at four levels of concentration (539 μg/kg, 140 μg/kg, 23 μg/kg and <2 μg/kg). The patulin content changes during the processing were followed. The samples were taken after the homogenization, pulping, pasteurization and aseptic packaging. All operations of apple puree production contributed to patulin reduction. The principle operation was pulping, where the patulin levels were reduced from 29% to 80% of original content.     

High hydrostatic pressure assisted degradation of patulin in fruit and vegetable juice blends

The degradation of patulin introduced into different juices then treated with high hydrostatic pressure (HHP) was evaluated. A model juice prepared from apple and spinach (AS) was studied along with commercially available apple-based beverages; Pineapple:Apple:Mint (PAM), Apple:Carrot:Beet:Lemon:Ginger (CAB) and Romaine:Celery:Cucumber:Apple:Spinach:Kale, Parsley:lemon (GJ). The extent of patulin degradation was found to be dependent on applied pressure and processing time (degradation rates ranged from 0.04 to 0.19 ppb/s). The extent of patulin degradation could also be significantly (P < 0.05) correlated with the sulfhydryl group concentration of the juice with ascorbic acid and nitrite being less significant. HHP treatment of 600 MPa for 300 s at 11 °C resulted in a 62 ppb decrease in patulin introduced into GJ juice which also contained the highest level of thiols (97 μM). The thiol concentration of the other juices ranged between 39 and 69 μM with a corresponding decrease in patulin of 43–49 ppb following the same HHP treatment. The study has illustrated that HHP can be applied as a risk management tool to control patulin in apple based beverages although the extent of mycotoxin degradation is dependent on processing conditions and composition of the juice.     

Determination of patulin in solid and semisolid apple and pear products marketed in Argentina

Patulin is an important index of quality and several methods are described for apple juice, but there is a lack of information about other products. The aim of this study was to implement an analytical method for solid and semisolid apple and pear products marketed in Argentina to evaluate patulin contamination. This method used a depectinization step and rapid clean-up with (SPE) Multisep®228 AflaPat multifunctional columns.The results showed that 21.6% of 51 products were contaminated (range 17–221 μg/kg, average of positive samples 61.7 μg/kg), but the highest levels were found in apple puree with 50% contaminated samples (average of positive samples 123 μg/kg). Efforts to decrease patulin on apple puree should be made.     

Application of pulsed light to patulin reduction in McIlvaine buffer and apple products

Numerous studies around the world have reported the occurrence of patulin in commercialized apple products. The persistence of this mycotoxin in apple products indicates that current methods used to reduce it during the manufacturing process are not entirely successful and reflects the need to evaluate new detoxification methods. The purpose of the present study was to investigate the effect of pulsed light (PL) dose on patulin degradation in McIlvaine buffer, apple juice and apple purée. The exposure of all samples to PL doses between 2.4 and 35.8 J/cm² resulted in a significant decrease in patulin levels. Patulin reduction in McIlvaine buffer did not depend markedly on the initial concentration of the mycotoxin. At the maximum dose tested, the remaining average patulin level dissolved in McIlvaine buffer was approximately 5–15%, while in apple juice the values declined up to 22%. In apple purée naturally contaminated with 29 μg/kg of patulin, exposure to a PL dose of 12 J/cm² provoked a 51% reduction in patulin concentration, while no residual contamination was detected for higher irradiation times.These results suggested that PL treatment would be a potential alternative method to reduce patulin contamination in apple products. However, further investigations need to be conducted to evaluate toxicological safety of patulin degradation product(s).     

Incidence and level of patulin contamination in pure and mixed apple juices marketed in Italy

A survey on the occurrence of patulin was conducted during 2005 on commercial pure apple juices (53 samples) and mixed apple juices (82 samples) marketed in Italy. The current study was undertaken to investigate the possible influence of the agro-food production process employed (conventional or organic), of the fruit percentage in the commercial product (higher or lower than 50%) and of the type of apple juice (clear or cloudy) on the occurrence and level of patulin contamination. Patulin could be quantified in 34.8% of the samples ranging from 1.58 to 55.41 μg kg⁻¹. With the exception of one sample, the level of patulin was lower than 50 μg kg⁻¹, the maximum permitted threshold in fruit juices according to the European legislation. Mean levels of patulin were significantly lower in mixed apple juices (4.54 μg kg⁻¹) than in pure apple juices (9.32 μg kg⁻¹). Levels of patulin contamination were comparable in clear and cloudy juices. A similar incidence of positive samples was found in conventional and organic apple based juices, and the magnitude between the mean contamination levels, although higher in organic (10.92 μg kg⁻¹) than in conventional juices (4.77 μg kg⁻¹), was not statistically significant (p = 0.771; Mann–Whitney test). The magnitude between the means of patulin contamination in juices containing more than 50% fruit (11.26 μg kg⁻¹) and in juices with 50% or less fruit (3.35 μg kg⁻¹) was statistically significant (p = 0.016; Mann–Whitney test).     

Control of Alicyclobacillus acidoterrestris in apple juice by citrus extracts and a mild heat-treatment

This paper reports on the use of different citrus extracts (biocitro, lemon extract and neroli) for the inhibition of the spores of two strains of Alicyclobacillus acidoterrestris; the research included 3 steps. First, the bioactivity of citrus extracts was assessed under in vitro conditions using a modified micro-dilution method; the concentration of extracts was from 20 to 500 ppm. Then, biocitro and lemon extract (0–80 ppm) were combined with two different thermal treatments (at 70 and 80 °C for 3–6 min) through a fractional factorial design and finally a treatment at 80 °C for 6 min was combined with either biocitro (80 ppm) or lemon extract (80 ppm) and tested in apple juice. Biocitro and lemon extract showed MIC values (Minimal Inhibitory Concentration) from 160 to 500 ppm; their effect could be enhanced by a thermal treatment at 80 °C for 3–6 min, both under in vitro conditions and in apple juice. Finally, biocitro or lemon extract could be added before and after the thermal treatment without any significant loss of their bioactivity.     

Control of Alicyclobacillus spp. vegetative cells and spores in apple juice with rosemary extracts

In this study, we initially investigated the antimicrobial activities of two commercial rosemary extracts (V20, V40) against Alicyclobacillus strains, with the ultimate purpose to determine whether these can be used in apple juice for the control of growth of alicyclobacilli. Minimum inhibitory concentrations (MICs) were first determined, and put through sensory analysis. Addition of the rosemary extracts to apple juice at their MICs did not change the colour, odour, taste or opacity of the apple juice. Growth kinetics studies with these rosemary extracts indicated a reduction in vegetative cells for Alicyclobacillus acidoterrestris, Alicyclobacillus hesperidum and Alicyclobacillus cycloheptanicus in Bacillus acidoterrestris broth and in apple juice. Further studies with A. acidoterrestris spores showed that the MICs of these rosemary extracts had relatively low effects on spore numbers in B. acidoterrestris broth, but had a spore number inhibition index >15% in apple juice. A four-fold increase in the rosemary extract concentrations showed the opposite effects: greater reduction in spores in B. acidoterrestris broth (inhibition index, >60%) than in apple juice (inhibition index, <10%). These data indicate that at their MICs, the V20 and V40 rosemary extracts allow outgrowth of spores but reduce vegetative cells, acting together with the low pH or other particular constituents of the apple juice. Rosemary extracts applied at their MICs thus represent an alternative method for the control of A. acidoterrestris in apple juice.     

Influence of processing parameters on the pulsed-light inactivation of Penicillium expansum in apple juice

The objective of this study was to evaluate the efficiency of pulsed light treatments on the inactivation of Penicillium expansum inoculated in apple juice. Different critical processing parameters, namely fluence (0.2 and 0.4 J/cm² per pulse), number of pulses (5, 10, 15, 20, 30 and 40 flashes applied from opposite product sides), depth of the juice layer (6, 8, 10 mm) and inoculation level (2.3 × 10⁴ CFU/mL and 3 × 10⁵ CFU/mL) were studied regarding their effect on microbial inactivation. Moulds inactivation increased with increasing number of pulses and fluence. Treatments led to microbial reductions of up to 3.76 log CFU/mL. Inactivation levels achieved with treatments performed on thin layers of juice (6 mm) were substantially higher than those observed after applying the same treatments on deeper layers of 8 or 10 mm. As well, P. expansum inactivation in juice samples with lower initial counts was greater than in samples with higher counts. Minor changes were noticed after PL treatments regarding juice pH and soluble solids; however, apple juice colour slightly darkened after applying and overall fluence of 32 J/cm² (40 pulses of 0.4 J/cm² from both upper and bottom sides). The obtained results suggest that PL-technology can be applied to efficiently reduce P. expansum counts in apple juice while maintaining the product quality.     

Solid-phase extraction of staphylococcal enterotoxin A in dairy products using an ion exchange resin

In this study, we developed a solid-phase extraction method for staphylococcal enterotoxin (SE), especially SE type A (SEA), in milk and dairy products. Ion-exchange resin, a cation-exchanger, was applied for the extraction of SEA, which was then measured using a sensitive fluorescence immunoassay. First, the optimal pH for the binding of SEA to the cation exchanger was determined to be pH 4.0. Second, the optimal volume of elution buffer, consisting of phosphate buffer with 0.5 M sodium chloride at pH 8.0, was estimated to be 6 ml for a column containing 4 ml wet volume of the cation-exchanger. Food samples (10 ml each) were then studied for the application of the extraction method. High recovery of SEA was obtained from raw milk (approximately 90%), sterilized milk product (approximately 60%), and skimmed milk powder (approximately 60%), whereas the recovery from cheese (approximately 40%) was lower than from other samples. These recoveries were almost constant at different concentrations of spiked SEA. The minimum measurable level with this extraction method was very low, being 0.25 ng/g in skimmed milk powder for example. With a larger volume (60 ml) of sample, this level was as low as 0.025 ng/g SEA in skimmed milk powder. The present extraction method was very simple and time saving in comparison with the conventional method with chloroform and trichloroacetic acid. The results of this study indicate that this extraction method could be applicable for the measurement of SEA in milk and dairy products.