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.     

Ability of inactivated yeast powder to adsorb patulin from apple juice

This study aimed to investigate the adsorption of patulin from apple juice, using two types of inactivated yeast powder: laboratory-prepared yeast powder (LYP) and commercial yeast powder (CYP). The effects of incubation time, pH, incubation temperature, adsorbent amount, and initial concentration of patulin and the stability of the yeast-mycotoxin complex were assessed. The results showed that the efficiencies of the two yeast types in adsorbing patulin were similar. The ability of the powders to remove patulin increased with longer incubation times, and patulin concentration was below detectable levels with LYP and CYP at approximately 36 and 30 h, respectively. The highest removal of patulin was achieved at pH 5.0 for both powder types, and there were no significant differences in patulin decrease at different temperatures (4, 29, and 37°C). Additionally, the adsorption percentage of patulin increased significantly with the increase of absorbent amount and decrease of initial concentration of patulin. Stability of the yeast-patulin complex was assessed, and patulin was more stable when washed in phosphate-buffered saline (pH 4.0) than in absolute ethyl alcohol. These results suggest that inactivated yeast powder has potential as a novel and promising adsorbent to bind patulin effectively.     

Patulin reduction in apple juice from concentrate by UV radiation and comparison of kinetic degradation models between apple juice and apple cider

Patulin, a mycotoxin produced by several genera of fungi, including Byssochlamys, Aspergillus, and Penicillium, has been an important concern in apple cider and apple juice due to its toxicity and health consequences. In this study, the effects of UV on the patulin level, physical and chemical properties, and sensory attributes in apple juice from concentrate were investigated. Kinetic modeling of patulin reduction by UV radiation in apple juice from concentrate was calculated and compared with the degradation rate observed previously in apple cider. From an initial patulin contamination of approximately 1,000 ppb (μg/liter), the UV exposure, ranging from 14.2 mJ/cm(2) (one pass) to 99.4 mJ/cm(2) (seven passes), was successful in reducing patulin levels by 72.57% ± 2.76% to 5.14% ± 0.70%, respectively. Patulin reduction by UV radiation followed first-order kinetic modeling in a fashion similar to first-order microbial inactivation. An exponential correlation between UV exposure and the percentage of patulin remaining was observed, giving an r(2) value of 0.9950. Apple juice was repeatedly exposed to 14.2 mJ/cm(2) for each treatment, and patulin levels were significantly decreased when compared with the level obtained with the previous UV exposure treatment. While there were no significant differences in the percentages of titratable acidity and ascorbic acid (P > 0.05), there were minor yet random sampling differences in pH and degrees Brix (1 °Brix is 1 g of sucrose in 100 g of solution; the °Brix represents the soluble solids content of the solution as percentage by weight [%, wt/wt]) (P ≤ 0.05). A significant difference (P ≤ 0.05) in sensory perception for the finished apple juice was detected between the control and the full seven-pass UV radiation treatment using an experienced consumer panel and a triangle test. Patulin reduction by UV radiation from both the current study and a previous study involving apple cider was compared, which showed that both matrices strongly fit a first-order kinetic degradation model. However, the kinetic constant for degradation in apple juice was approximately 5.5 times greater than that observed in an apple cider matrix.     

CONTAMINATION OF PATULIN IN CLEAR APPLE JUICE IN MASHHAD, IRAN

Patulin is a frequent contaminant of moldy and rotten apples and apple products. The aim of this study was to evaluate patulin contamination in 58 apple juices collected from a retail market in Mashhad during winter and spring of 2006.
Samples were assayed for patulin by high-performance liquid chromatography. Fifty-four samples were positive for patulin at levels that ranged from 10.5 to 121.8 µg/L, and six samples had patulin levels higher than 50 µg/L. The overall mean of patulin concentration was 29.2  ±  19.5 µg/L. Forty-eight samples had patulin concentration between 5 and 50 µg/L. Although the mean concentration of patulin samples was lower than Iranian maximum tolerated level of 50 µg/L, contamination of 10% of the samples at levels higher than 50 µg/L indicated the need for improving production techniques by the industry.

PRACTICAL APPLICATIONS

Studies have shown that the concentration of patulin may exceed the determined limits in apple juice and in other fruit products. Its presence can be a potential threat to the health of consumers, particularly children. The results may help us in understanding what should be the level of patulin in apple juice. The awareness of the apple industry of patulin contamination in fruit and the implementation of improved techniques for the production of apple products with reduced patulin concentrations have contributed to the quality of apple juice that are available on the Iranian market.     

利用Saccharomyces cerevisiae KD控制苹果汁中棒曲霉素的污染

研究一株食品生产用酿酒酵母Saccharomyces cerevisiae KD在培养基及市售100%苹果汁中对棒曲霉素污染的控制作用。通过高效液相色谱法对棒曲霉素进行定量,分析起始棒曲霉素浓度、菌体接种量和培养基pH对S. cerevisiae KD去除棒曲霉素活力的影响;利用酶标仪监测S. cerevisiae KD的生长状况,且通过检测可溶性固形物、酸度、总酚、黄酮含量对菌体发酵后苹果汁的品质进行了评估。结果表明:有氧条件下S. cerevisiae KD能够在28 h内完全去除培养基中的棒曲霉素,其去除机理包括物理吸附和酶解;在较低的起始棒曲霉素浓度和较高的菌体接种量条件下,S. cerevisiae KD对棒曲霉素的去除率较高,但在培养后期,不同菌体接种量下棒曲霉素的去除率接近一致;实验还发现酸性条件有利于S. cerevisiae KD去除棒曲霉素。此外,S. cerevisiae KD对棒曲霉素的耐受性较强,甚至在棒曲霉素浓度高达100 mg/L的环境中依然能较好生长。在市售100%苹果汁中,S. cerevisiae KD也能高效控制棒曲霉素的污染,且与Lactococcus lactis MG1363联合发酵2 d后,果汁中已无棒曲霉素检出,总酚含量显著高于发酵前苹果汁(p<0.05),发酵果汁的品质较好。结论:S. cerevisiae KD可有效控制食品中棒曲霉素的污染,具有潜在的应用前景。     

A limited survey of retail apple and grape juices for the mycotoxin patulin

A retail survey (38 samples) of apple, grape and other juice-based products for the mycotoxin patulin has been carried out using HPLC analysis. Positive results were confirmed by GC-MS of the TMS derivative using chemical ionization selected ion monitoring. No grape juice or mixed juice product contained detectable patulin (limit 5 micrograms/l), nor did the majority (58%) of the apple juices. Low levels of patulin (in the range 5-10 micrograms/l) were present in six apple juices and the remaining four positive samples contained 16, 18, 30 and 56 micrograms/l.     

Potential of two Metschnikowia pulcherrima (yeast) strains for in vitro biodegradation of patulin

Patulin contamination of apple and other fruit-based foods and beverages is an important food safety issue, as consumption of these commodities throughout the world is great. Studies are therefore necessary to reduce patulin levels to acceptable limits or undetectable levels to minimize toxicity. This study was undertaken to investigate the efficacy of two Metschnikowia pulcherrima strains (MACH1 and GS9) on biodegradation of patulin under in vitro conditions. These yeast strains were tested for their abilities to degrade patulin in liquid medium amended with 5, 7.5, 10, and 15 μg/ml patulin and a yeast cell concentration of 1 × 10(8) cells per ml at 25°C. Of the two strains tested, MACH1 completely (100%) reduced patulin levels within 48 h, and GS9 within 72 h, at all concentrations of patulin. MACH1 effectively degraded the patulin within 24 h by 83 to 87.4%, and GS9 by 73 to 75.6% at 48 h, regardless of concentration. Patulin was not detected in yeast cell walls. This indicates that yeast cell walls did not absorb patulin, and that they completely degraded the toxin. Patulin had no influence on yeast cell concentration during growth. Therefore, these yeast strains could potentially be used for the reduction of patulin in naturally contaminated fruit juices. To our knowledge, this is the first report regarding the potential of M. pulcherrima strains for patulin biodegradation.     

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.     

Evaluation of several culture media for production of patulin by Penicillium species

The aim of this study was to evaluate different species of Penicillium to identify those which have the potential to produce the greatest amount of the mycotoxin, patulin. Additionally, six different culture media were compared to determine maximum patulin production. Eleven different strains of Penicillium species were selected because they had previously been reported to be producers of patulin. The strains included Penicillium expansum, Penicillium griseofulvum (formerly Penicillium urticae), Penicillium clavigerum, and Penicillium coprobium and a recent Penicillium sp. isolated from an apple. Cultures were grown in duplicate in three different liquid media: potato dextrose, malt extract, and glucose/yeast extract/peptone, both with and without manganese supplementation. Patulin production was compared at 24, 48, 72, and 96 h. Variability in patulin production occurred among the different species, growth media used, and time of incubation. All three of the P. griseofulvum isolates were the highest producers of patulin at 96 h. For most of the strains, potato dextrose broth supplemented with manganese was optimal for maximum production of patulin. Although P. expansum is frequently cited as the most likely source of patulin in apple juice, certain other Penicillium species are capable of producing more patulin than strains of P. expansum. The apple juice industry should be alert to the possibility that Penicillium species other than P. expansum can be responsible for the occurrence of patulin.     

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     

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     

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.     

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.     

Comprehensive Review of Patulin Control Methods in Foods

The mycotoxin, patulin (4‐hydroxy‐4H‐furo [3, 2c] pyran‐2[6H]‐one), is produced by a number of fungi common to fruit‐ and vegetable‐based products, most notably apples. Despite patulin's original discovery as an antibiotic, it has come under heavy scrutiny for its potential negative health effects. Studies investigating these health effects have proved inconclusive, but there is little doubt as to the potential danger inherent in the contamination of food products by patulin. The danger posed by patulin necessitates its control and removal from foods products, creating a demand for handling and processing techniques capable of doing so, preferably at low cost to industry. With this being the case, much research has been devoted to understanding the basic chemical and biological nature of patulin, as well as its interaction within foods and food production. While past resarch has elucidated a great deal, patulin contamination continues to be a challenge for athe food industry. Here, we review in depth the past research on patulin with an emphasis upon its influence within the food industry, including its regulation, health effects, biosynthesis, detection, quantification, distribution within foods, and control, during the various stages of apple juice production. Finally, key areas where future patulin research should focus to best control the patulin contamination problem within the food industry are addressed.     

Analysis of patulin in apple products by liquid–liquid extraction,solid phase extraction and matrix solid-phase dispersion methods: a comparative study

Patulin is a marker of quality in the apple and apple juice industry and due to the potential risk for human health, reliable and potential methods for extracting patulin from a sample are therefore needed. In this study, the three methods with liquid–liquid extraction, matrix solid-phase dispersion (MSPD) and with solid-phase extraction (SPE) were studied for extracting patulin from different apple products. Result showed that for AJC and apple sample, MSPD method is most suitable for extracting patulin among the three methods. The recovery rates of AJC and apple sample were 80.35–114.46 and 79.68–94.32%, respectively, the coefficient variations were 3.18–4.90%; For dilute juice, SPE procedure is suitable for analysis of patulin and the recovery rates were and 85.35–90.14%.     

Inter-laboratory study for validation of a Japanese official analytical method for determination of patulin in apple juice

To validate a modified version of AOAC official method of analysis 995.10 as an official standard in Japan for determination of patulin in apple juice, an inter-laboratory study was performed in 11 laboratories using a non-contaminated sample, 2 naturally contaminated samples and 2 spiked samples of apple juice. For naturally contaminated apple juices, the relative standard deviations for repeatability and reproducibility were 3.2, 7.1% and 10.0, 21.7%, respectively. HORRAT values were 0.4, 0.9. The average recovery of patulin from spiked sample was 83.7%. The limit of quantification was calculated as 10 microg/kg. From these results, the method was thought to be suitable as an official standard for determination of patulin in apple juice in Japan.     

Investigation of patulin contamination in apple juice sold in retail outlets in Italy and South Africa

A study of apple juice products sold in Italy and South Africa was initially carried out on 20 samples bought in Cesena, Italy, and Tygerberg in Cape Town, South Africa. The samples were bought at random and analysed for patulin contamination. All 12 of the Italian samples had no detectable levels of patulin, except one, which was just slightly above the lowest regulatory limit of 10 ng ml^-1. On the other hand, five of the eight South African samples were all contaminated with patulin levels above 10 ng ml^-1, with one showing a concentration of 75 ng ml^-1, well above the highest regulated limit of 50 ng ml^-1. This latter result led to a more targeted investigation with 14 samples being purchased in the low-income areas of Tygerberg where the initial samples were sourced. These samples confirmed that there might be a problem of mycotoxin contamination in apple juices products sold to low-income consumers because half of the samples showed patulin contamination of which four had levels well above the acceptable limits. This is the first study in South Africa to look at apple juice products in low-income areas and it points to a need to intervene and introduce quality systems in the supply chain of the manufacture and packaging of apple juice products by independent small business.     

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。     

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.     

EFFECT OF COMMERCIAL PROCESSING STAGES OF APPLE JUICE ON PATULIN, FUMARIC ACID AND HYDROXYMETHYLFURFURAL (HMF) LEVELS

The effects of different stages of commercial apple juice production on the patulin, fumaric acid and hydroxymethylfurfural (HMF) contents of apple juice were investigated. Heat treatment and activated charcoal were effective for the reduction of patulin. The average reduction of patulin were 13.4% and 22.9% for heat treatment and activated charcoal, respectively. Statistical analysis showed no significant differences (P<0.05) in the presence of fumaric acid between different treatments. Filtration and heat treatment caused an apparent increase in the HMF content, while activated charcoal caused a decrease. The average increments of HMF were 40.4% and 38.0% for separation and heat treatment, respectively, while the average reduction of HMF was 23.7% for activated charcoal. Spiking of samples with known amounts of patulin and fumaric acid revealed respective recovery rates of 96.4% and 95.9%.