The reduction of patulin in apple juice by three different types of activated carbon

Three types of activated carbon (NORIT SA 4, NORIT SX 4 and NORIT CA 1) were investigated for their ability to reduce patulin levels in apple juice at various Brix levels and temperatures. The steamactivated carbons (NORIT SA 4 and NORIT SX 4) exhibited similar adsorption isotherms at a dosage level of 1g/l. They achieved patulin reductions of 80% and 70% respectively in 12° Brix juice at 55°C. The similarity in performance between the steam-activated carbons implies that the purity and the surface acidity does not influence the adsorption of patulin. Chemically-activated carbon (NORIT CA 1) was less effective in removing patulin and achieved only a 45% reduction at a dose of 1g/l. Patulin removal was influenced by juice Brix in that higher carbon doses were required at higher Brix levels for equivalent removal efficiency. At a dose of 1g/l, NORIT SA 4 removed only 45% patulin from a 20° Brix juice. The removal of patulin from either 12 or 20° Brix juice by NORIT SA 4 at 1g/l was not influenced by temperature changes in the range 30 to 65°C.     

Long-term survey of patulin in apple juice concentrates produced in Turkey.

A liquid chromatographic method described by us elsewhere was evaluated for a long-term survey of patulin in apple juice concentrates. Patulin was separated on a reversed phase C18 LC column with water-acetonitrile (99:1) as the mobile phase and quantitated with a photodiode array (PDA) detector. Relatively low amounts of patulin (< 5 micrograms/l for single strength juice at 11.2 degrees Bx) were detected in apple juice concentrates and confirmed by PDA detector, comparing the corresponding UV spectra with that of patulin standard. Four hundred and eighty two apple juice concentrates produced through 1996-99 were analysed for their patulin contents. Year-to-year variations in patulin levels of apple juice concentrates were found out to be statistically significant. Patulin contamination levels of apple juice concentrates tended to decrease through the years and averaged 63, 43, 19 and 31 micrograms/l in 1996, 1997, 1998 and 1999, respectively. Percentages of concentrates exceeding the maximum permitted concentration of 50 micrograms/l were 52%, 34%, 8% and 8% for 1996, 1997, 1998 and 1999, respectively.     

Effect of heat treatment and evaporation on patulin and some other properties of apple juice

Patulin, hydroxymethylfurfural (HMF), colour and clarity values of 20 apple juice samples with different Brix contents were determined after heat treatment (90 and 100 °C for 5, 10, 15 and 20 min under atmospheric pressure) and evaporation (70 and 80 °C for 5, 10, 15 and 20 min) processes. As the heating and evaporation times increased, the concentration of patulin in the apple juice samples decreased. The 90 and 100 °C heat treatments resulted in reductions in patulin concentration of 18.81 and 25.99% respectively after 20 min; the corresponding values were 9.40 and 14.06% for 70 and 80 °C evaporation respectively. The increase in HMF was higher with heat treatment than with evaporation. Evaporation decreased the colour values of the samples, whereas heat treatment increased them. © 2003 Society of Chemical Industry     

Apple quality,storage, and washing treatments affect patulin levels in apple cider

Patulin is a mycotoxin produced primarily by Penicillium expansum, a mold responsible for rot in apples and other fruits. The growth of this fungus and the production of patulin are common in fruit that has been damaged. However, patulin can be detected in visibly sound fruit. The purpose of this project was to determine how apple quality, storage, and washing treatments affect patulin levels in apple cider. Patulin was not detected in cider pressed from fresh tree-picked apples (seven cultivars) but was found at levels of 40.2 to 374 microg/liter in cider pressed from four cultivars of fresh ground-harvested (dropped) apples. Patulin was not detected in cider pressed from culled tree-picked apples stored for 4 to 6 weeks at 0 to 2 degrees C but was found at levels of 0.97 to 64.0 microg/liter in cider pressed from unculled fruit stored under the same conditions. Cider from controlled-atmosphere-stored apples that were culled before pressing contained 0 to 15.1 microg of patulin per liter, while cider made from unculled fruit contained 59.9 to 120.5 microg of patulin per liter. The washing of ground-harvested apples before pressing reduced patulin levels in cider by 10 to 100%, depending on the initial patulin levels and the type of wash solution used. These results indicate that patulin is a good indicator of the quality of the apples used to manufacture cider. The avoidance of ground-harvested apples and the careful culling of apples before pressing are good methods for reducing patulin levels in cider.     

Reduction of patulin during apple juice clarification

Patulin is a mycotoxin produced by a number of molds involved in fruit spoilage. This compound is carcinogenic and teratogenic. Various methods are currently used to reduce the levels of patulin in apple juice, namely, charcoal treatment, chemical preservation (sulfur dioxide), gamma irradiation, fermentation, and trimming of fungus-infected apples. Many of these processes are expensive and time-consuming. Therefore, there is a need to find a convenient and economical process to control patulin levels. This study was undertaken to evaluate the effectiveness of several clarification processes for the reduction of patulin. Clarification was carried out on a laboratory scale. Apple pulp was spiked with patulin, pressed, and clarified using four different processes, namely, fining with bentonite, enzyme (pectinase) treatment, paper filtration, and centrifugation. Patulin was recovered from the clarified juice by liquid-liquid extraction, and solid-phase chromatography was used for sample clean-up prior to analysis by high-performance liquid chromatography (HPLC). The minimum detectable limit using HPLC was 20 microg/liter. Pressing followed by centrifugation resulted in an average toxin reduction of 89%. Total toxin reduction using filtration, enzyme treatment, and fining were 70, 73, and 77%, respectively. Patulin reduction was due to the binding of the toxin to solid substrates that was verified by analyzing the clarified juice as well as the filter cake, pellet, and sediment. The combined concentrations correlated to the spiked concentration. These results reveal that clarification was successful in the reduction of patulin levels in apple juice. However, clarification resulted in high levels of patulin in the pressed pulp after filtration and centrifugation, and this could be harmful if they are used as animal feeds.     

Incidence of patulin in apple juices marketed in Turkey.

The purpose of this study was to investigate the patulin contamination of apple juices consumed by the Turkish population. Patulin was detected using high-performance liquid chromatography (HPLC) with a UV detector at 280 nm, and the identification of patulin was further confirmed by thin-layer chromatography (TLC). Using HPLC, the recoveries were 79.9 +/- 6.7% and 83.7 +/- 4.6%, and the coefficients of variation were 8.4 and 5.5% for apple juices spiked with the known amounts of patulin (60 and 120 microg/liter. respectively). The minimum patulin level detected was 5 ng in a standard solution and 5 microg/liter in apple juices. The TLC method was used only to confirm patulin levels higher than 20 microg/liter (100 ng/spot) in apple juices. The total number of samples was 45. Patulin was present in detectable levels in 60% of apple juices at concentrations ranging from 19.1 to 732.8 microg/liter. Forty-four percent of the apple juice samples had patulin contamination levels higher than 50 microg/ liter, which is the allowable upper limit in Turkey.     

Fate of patulin in the presence of the yeast Saccharomyces cerevisiae

Patulin is known to become analytically non-detectable during the production of cider from contaminated apple juice. The fate of [¹⁴C]-labelled patulin during the alcoholic fermentation of apple juice was studied. Three commercial cider strains of Saccharomyces cerevisiae degraded patulin during active fermentative growth, but not when growing aerobically. The products of patulin degradation were more polar than patulin itself and remained in the clarified fermented cider. Patulin did not appear to bind to yeast cells or apple juice sediment in these model experiments. HPLC analysis of patulin-spiked fermentations showed the appearance of two major metabolites, one of which corresponded by both TLC and HPLC to E-ascladiol prepared by the chemical reduction of patulin using sodium borohydride. Using a diode array detector, both metabolites had a λ_max = 271nm, identical to that of ascladiol. Thenmr spectrum of a crude preparation of these metabolites showed signals corresponding to those of the E-ascladiol prepared chemically and a weaker set of signals corresponding to those reported in the literature for Z-ascladiol.     

Occurrence of patulin in organic, conventional, and handcrafted apple juices marketed in Belgium

The aim of this research was to compare the occurrence of patulin in a large group of organic, conventional, and handcrafted apple juices marketed in Belgium. An analytical procedure based on high-performance liquid chromatography with UV detection was validated and used to analyze 177 apple juice samples: 65 organic, 90 conventional, and 22 handcrafted. Patulin was detected in 22 samples (12%), and quantification was possible in 10 (6%) of these samples. The patulin content was higher than the European legal limit of 50 microg/liter in two samples of organic apple juice. Although, the incidence of patulin in organic (12%), conventional (13%), and handcrafted (10%) apple juices was not significantly different (P = 0.863), the mean concentration of patulin in contaminated samples was significantly higher in organic (43.1 microg/liter) than in conventional (10.2 microg/liter) (P = 0.02) and handcrafted (10.5 microg/liter) (P = 0.037) apple juice. The highest patulin concentrations were found in the most expensive apple juices because of the higher price of organic apple juice. This relation was not observed when only conventional apple juices were analyzed.     

Reducing patulin contamination in apple juice by using inactive yeast

The mycotoxin, patulin (4-hydroxy-4H-furo[3,2c]pyran-2[6H]-one), is a secondary metabolite produced mainly in rotten parts of fruits and vegetables, most notably apples and apple products, by a wide range of fungal species in the genera Penicillium, Aspergillus, and Byssochlamys. Due to its mutagenic and teratogenic nature and possible health risks to consumers, many countries have regulations to reduce levels of patulin in apple products. In the present study, reduction of patulin contamination in apple juice by using 10 different inactivated yeast strains was assessed. Our results indicated that nearly twofold differences in biomass existed among the 10 yeast strains. Eight of the 10 inactivated yeast strains could provide >50% patulin reduction in apple juice within 24 h, with the highest reduction rate being >72%. Furthermore, juice quality parameters, i.e., degrees Brix, total sugar, titratable acidity, color value, and clarity, of the treated apple juice were very similar to those of the untreated patulin-free juice. Potential applications of using inactivated yeast strain for patulin control are also discussed.     

Survey of patulin occurrence in apple juice and apple products in Catalonia,Spain, and an estimate of dietary intake

This study was conducted to assess patulin exposure in the Catalonian population. Patulin levels were determined in 161 apple juice samples, 77 solid apple-based food samples and 146 apple-based baby food samples obtained from six hypermarkets and supermarkets from twelve main cities of Catalonia, Spain. Patulin was analysed by a well-established validated method involving ethyl acetate extraction and direct analysis by high-performance liquid chromatography (HPLC) with ultraviolet light detection. Mean patulin levels for positive samples in apple juice, solid apple-based food and apple-based baby food were 8.05, 13.54 and 7.12 µg kg^?1, respectively. No samples exceeded the maximum permitted levels established by European Union regulation. Dietary intake was separately assessed for babies, infants and adults through a Food Frequency Questionnaire developed from 1056 individuals from Catalonia. Babies were the main group exposed to patulin, however no risk was detected at these levels of contamination. Adults and infants consumers were far from risk levels. Another approach to determine estimated exposure was conducted through Monte Carlo simulation that distinguishes variability in exposures from uncertainty of distributional parameter estimates.     

Development and characterization of a carbon-based composite material for reducing patulin levels in apple juice

Patulin, a heterocyclic lactone produced by various species of Penicillium and Aspergillus fungi, is often detected in apple juices and ciders. Previous research has shown the effectiveness of granular activated carbon for reducing patulin levels in aqueous solutions, apple juices, and ciders. In this study, ultrafine activated carbon was bonded onto granular quartz to produce a composite carbon adsorbent (CCA) with a high carbonaceous surface area, good bed porosity, and increased bulk density. CCA in fixed-bed adsorption columns was evaluated for efficacy in reducing patulin levels from aqueous solutions and apple juice. Columns containing 1.0, 0.5, and 0.25 g of CCA were continuously loaded with a patulin solution (10 microg/ml) and eluted at a flow rate of 1 ml/min. Results indicated that 50% breakthrough capacities for patulin on 1.0-, 0.5-, and 0.25-g CCA columns were 137.5, 38.5, and 19.9 microg, respectively. The effectiveness of CCA to adsorb patulin and prevent toxic effects was confirmed in vitro using adult hydra in culture. Hydra were sensitive to the effects of patulin, with a minimal affective concentration equal to 0.7 microg/ml; CCA adsorption prevented patulin toxicity until 76% breakthrough capacity was achieved. Fixed-bed adsorption with 1.0 g of CCA was also effective in reducing patulin concentrations (20 microg/liter) in a naturally contaminated apple juice, and breakthrough capacities were shown to increase with temperature. Additionally, CCA offered a higher initial breakthrough capacity than pelleted activated carbon when compared in parallel experiments. This study suggests that CCA used in fixed-bed adsorption systems effectively reduced patulin levels in both aqueous solutions and naturally contaminated apple juice; however, the appearance and taste of apple juice may be affected by the treatment process.     

A quantification and confirmation method of patulin in apple juice by GC/MS

A sensitive and selective method for quantification and confirmation of patulin in apple juice by GC/MS was developed. By this method, patulin was precisely determined and confirmed down to the level of 1 and 5 microg/kg in samples, respectively. Patulin was extracted with ethyl acetate from a sample and then hexane was added to the concentrated extract solution. Significant amounts of insoluble impurities were filtered off, followed by further clean-up by solid-phase extraction with combined silica gel and Florisil cartridges. The filtration step in a low-polarity condition was very effective to remove the impurities in the sample extract solution. The eluate from the cartridges was evaporated to dryness under reduced pressure and patulin was determined and confirmed by GC/MS after derivatization with 2.5% N,O-bis(trimethylsilyl)trifluoroacetamide ethyl acetate solution. Patulin was determined in the selected ion monitoring mode (m/z 226) and confirmed in the SCAN mode (m/z 40-340). The recovery from apple juice spiked with 10-500 microg/kg ranged from 93.4 to 100%. The limits of detection and quantification were 0.1 (S/N = 3) and 1 microg/kg (S/N = 30) of patulin in samples, respectively. Levels down to 5 microg/kg of patulin in sample were readily confirmed.     

Detection of patulin in apple juices marketed in the Tohoku district, Japan

Patulin is a mycotoxin mainly produced by Penicillium and Aspergillus. We investigated the incidence of patulin contamination in 179 samples of apple juice and 9 samples of mixed juice (containing apple juice concentrate as an ingredient) commercially available in the Tohoku district of Japan. Patulin was detected in 3 of 143 samples containing domestic fruits and in 6 of 45 samples containing imported products and products produced in Japan using imported apple juice concentrate. Patulin analyses were carried out using high-pressure liquid chromatography with a detection limit of 4 microg/liter. The patulin content of contaminated domestic samples (three samples with concentrations ranging from 6 to 10 microg/liter), imported samples (one sample with a concentration of 15 microg/liter), and domestic samples produced containing imported concentrate (five samples with concentrations ranging from 6 to 9 microg/liter) was lower than the maximum limit of 50 microg/liter currently adopted by many countries, including Japan.     

Free and bound patulin in cloudy apple juice

During validation of an HPLC-UV method for patulin analysis, a time and concentration dependent recovery of patulin was observed. Spiked cloudy apple juice was analysed on successive days, which resulted in recoveries on day 3 which were up to 20% lower than on day 1. This reduction was caused by an interaction between patulin and the solid parts of cloudy apple juice. Since these solid parts are richer in proteins compared to the liquid phase of cloudy apple juice, and the binding of patulin to proteins has been described in the literature, patulin will most probably interact with the proteins in the solid parts. As a consequence, up to 20% of the present patulin is bound and not detected during HPLC-UV analysis, which can lead to an underestimation on toxicological level.     

浓缩苹果汁加工链中棒曲霉素的动态分析研究

研究了浓缩苹果汁加工过程中各工序对棒曲霉素含量的影响。结果表明,喷淋、拣选、清洗是去除棒曲霉素的关键步骤,去除率为60.18％;吸附树脂对棒曲霉素去除效果显著。此外,研究了加工季节中棒曲霉素的变化规律,为果汁加工厂家建立科学完善的HACCP管理体系提供了理论依据。     

Heat resistance of Escherichia coli O157:H7 in apple juice.

The objective was to determine the effect of cider composition on the heat resistance of Escherichia coli O157:H7. The average D52 value in a model Empire apple juice was 18 min with a z value of 4.8 degrees C. Increasing the Brix from 11.8 to 16.5 degrees had no effect on thermal resistance, while increasing L-malic acid from 0.2 to 0.8%, or reducing the pH from 4.4 to 3.6 sensitized the cells to heat. The greatest effect on heat resistance was afforded by the preservatives benzoic and sorbic acids: D50 values in ciders containing 1,000 mg/l were 5.2 min in the presence of sorbic acid and only 0.64 min in the presence of benzoic acid. Commercial apple juice concentrates yielded lower numbers of survivors than single-strength juices even though their higher sugar concentrations of about 46 degrees Brix increased heat resistance.     

Effect of thiamine hydrochloride,pyridoxine hydrochloride and calcium-d-pantothenate on the patulin content of apple juice concentrate

Thiamine hydrochloride, pyridoxine hydrochloride and calcium-d-pantothenate were applied apple juice concentrates (AJC) at various doses in order to reduce the patulin content. AJC samples containing high levels of patulin were stored at 22 +/- 2 degrees C and 4 degrees C for 6 months after vitamins were added. Patulin was fully degraded at the end of a 6-month period in samples stored at 22 +/- 2 degrees C, on the other hand, other quality parameters diminished significantly. Without any considerable reduction on other quality parameters, applications of 1000 and 2500 mg/kg calcium-d-pantothenate resulted in reduction of patulin of 73.6 and 94.3%, respectively, however, 42.1% of patulin reduction was observed in the control sample of AJC stored for 1 month at 22 +/- 2 degrees C. Addition of thiamine hydrochloride (1000 mg/kg), pyrodoxine hydrochloride (625 or 875 mg/kg) and calcium-d-pantothenate (1000 or 2500 mg/kg) into the samples and storage at 4 degrees C for 6 months yielded 55.5 to 67.7% of patulin reduction which was only 35.8% for the control while the other quality parameters were protected adequately.     

The effects of processing technology on the patulin content of juice during commercial apple juice concentrate production

 The effects of different treatments on the patulin content of apple juice during the production of industrial apple juice concentrate were investigated. Conventional clarification using a rotary vacuum precoat filter was found to be more effective than using ultrafiltration for the removal of patulin from apple juice. The average losses of patulin were 39% and 25% for conventional clarification and filtration, and ultrafiltration, respectively. Washing and handling appeared to be the most critical steps in removing patulin from apples since up to 54% could be removed using high-pressure water spraying. Received: 22 January 1998 / Revised version: 21 April 1998     

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.     

The effects of processing technology on the patulin content of juice during commercial apple juice concentrate production

 The effects of different treatments on the patulin content of apple juice during the production of industrial apple juice concentrate were investigated. Conventional clarification using a rotary vacuum precoat filter was found to be more effective than using ultrafiltration for the removal of patulin from apple juice. The average losses of patulin were 39% and 25% for conventional clarification and filtration, and ultrafiltration, respectively. Washing and handling appeared to be the most critical steps in removing patulin from apples since up to 54% could be removed using high-pressure water spraying. Received: 22 January 1998 / Revised version: 21 April 1998