Mycotoxin Prevention and Control in Agriculture - American Chemical

Chapter 5

Downloaded by AUBURN UNIV on December 26, 2017 | http://pubs.acs.org Publication Date (Web): December 20, 2009 | doi: 10.1021/bk-2009-1031.ch005

Physical protection of apple products from patulin contamination Seiji Bandoh1, Keisuke Higashihara1, Masahiko Takeuchi2, Katsumi Ohsawa3 and Tetsuhisa Goto1 1

Faculty of Agriculture, Shinshu University, Nagano, Japan. 2 Agricultural Technology Institute of Nagano Farmers’ Federation, Nagano, Japan, 3 Food Technology Department of Nagano Prefecture General Industrial Technology Center, Nagano, Japan.

Patulin is a mycotoxin produced by various Aspergillus and Penicillium fungi. Among these fungi, P. expansum often invades and damages apples and can cause patulin contamination in apple products. To prevent patulin contamination of apple products proper storage of apples before processing and physical removal of damaged portions of apples are very important. Spores of P. expansum were inoculated onto sound apples (mainly Fuji apples) and cultured. Decay caused by the growth of P. expansum and patulin content were measured. The movement of patulin within damaged apples was relatively restricted. Therefore patulin was effectively reduced in apples by removal of the physically damaged portion of the apples through either trimming or washing. However, P. expansum can grow and produce patulin on apples at relatively low temperatures, even at +1 °C. So for storage for longer than 3 months, apples need to be stored below 0 °C in order to prevent further production of patulin.

© 2009 American Chemical Society Appell et al.; Mycotoxin Prevention and Control in Agriculture ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

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Introduction Patulin is a mycotoxin produced by various Aspergillus, Penicillium and Byssochlamys fungi (1, 2). Among these fungi, P. expansum often invades and damages apples and can cause patulin contamination in apple products (1, 3, 4, 5). Patulin has antibiotic effects on both Gram negative and Gram positive bacteria (6). Patulin also has various toxic effects, including DNA damage (7), mutagenicity (8) and neurotoxicity (9). Therefore, Codex Alimentarius has recommended a patulin content of less than 50 μg/kg in apple products intended for human consumption and in many countries patulin regulations exist (10). Patulin can be partially removed from clear type apple juice during processing (11). However, patulin is relatively heat stable and tolerant to acidic conditions (12). Once P. expansum causes decay and produces patulin on apples, it is not so easy to reduce patulin contamination in product, such as straight (cloudy) type apple juice and puree. Therefore, to prevent patulin contamination of apple products both the proper storage of apples before processing and the physical removal of damaged portions of apples are very important.

Materials and Methods Chemicals and samples Patulin, 4-Hydroxy-4H-furo-[3,2-c]pyran-2(6H)-one, standard was purchased from Sigma-Aldrich (MO, USA) and Biopure (Tulln, Austria). Patulin standard stock solution (2 mg/mL) was made in ethyl acetate and stored at -20 °C. For the patulin working solution, an aliquot of patulin stock solution was taken into a small amber glass vial and dried under a gentle stream of N2 gas then dissolved with water which was ajusted its pH as 4 by acetic acid. 5hydroxymethylfurfural (5-HMF), was purchased from Acros (Geel, Belgium) and prepared same as patulin. All other chemicals were either HPLC grade or GR grade and purchased from Kanto Chemical Co. Ltd (Tokyo, Japan) and used without further purification. P. expansum used for this study was cultured on PDA (Potato Dextrose Agar) slant medium at 20 °C in the dark for 2 to 3 weeks. Preparation of mold damaged apple sample Spores of P. expansum were suspended in 0.1% Tween 20 solution (ca 106 C.F.U./mL). The surface of a sound apple was wiped with 80 % (v/v) ethanol and then spores were inoculated into the apple using a needle to about 1-2 mm below the surface. Apples were individually wrapped in aluminum foil then incubated at designated temperature in the dark. Temperature of incubators and apple samples during cultivation was monitored using a Center 309 data logger thermometer (MK Scientific, Tokyo, Japan). After cultivation, the diameter of the decayed area of apple was measured and then the apple was pressed to prepare juice for patulin analysis. Amounts of patulin in apple juices were

Appell et al.; Mycotoxin Prevention and Control in Agriculture ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

71 measured using the method dicussed below. For each study 5 to 6 apples were used. To study the relation between size of decay and patulin accumulation on apple, four strains of P. expansum were inoculated on four different cultivars of apple (Fuji, Ohrin, Yoko and Shinano gold) individually and cultured from +1 to 20 °C for 5 to 83 days then measued the size of visible decay and amount of patulin in the apple were measured.


Preparation of sample and extraction of patulin from apple juice For the study of the distribution of patulin in damaged apples, each apple was marked out into 36 sections. First the apple was divided into 9 sections using a commercially available apple cutter (vertically) then each piece of apple was divided into 4 sections (horizontally) (Figure 2). Ten Fuji apples were treated in the same way. The same sections from each apple were combined and then pressed to prepare the juice for analysis. Levels of patulin in apple juices were measured using AOAC-OMA method 995.10 (13, 14) with a minor modification. Also for some samples, a method (15) with a more extensive clean up was used for sample preparation. Prepared samples were dissolved in water ajusted its pH as 4 with acetic acid and analyzed by LC and/or LC-MS. Analysis of patulin. Patulin was analyzed by a single quadropole type LC-MS (Shimadzu LCMS-2010EV) with a UV detector. For quantitative purpose, UV absorption was mainly used and MS detection was mainly used for confirmation. The LC portion of the LCMS-2010EV system was a model LC-2010CHT liquid chromatograph system (Shimadzu). Patulin was separated on an ODS column (Synergi Hydro-RP 80À, 2.0 mm i.d. x 250 mm, Phenomenex) at 40 °C. The mobile phase was a mixture of water and acetonitrile (95:5, v/v) with a flow rate of 0.2 mL/min. Patulin was monitored by a UV absorbance detector set at 276 nm. Electrospray ionization (ESI) was used to introduce the sample to the MS and the patulin was detected at m/z 153, 125 and 109 by selected ion monitoring mode. ESI was carried out at a spray voltage of -3.5 kV, temperatures of 200 °C and 250 °C were applied to the Curved Desolvation Line (CDL) and heat block with nitrogen as a nebulizer gas at a flow rate of 1.5 L/min. Voltages of CDL and the detector were at 15.0 V and 1.5 kV. Samples were analyzed in negative ion mode.


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Results and Discussion LC and LC-MS analysis


As shown in Figure 1, patulin was detected at a retention time of about 13.5 min by both UV and MS and clearly separated from HMF and other materials. The lower detection limits of patulin by UV and LC-MS (m/z=153) were 0.01 and 0.1 ng, repectively. The calibration curves by UV and MS were linear from 0.05 to 40 ng and 0.5 to 40 ng, respectively. Patulin was successively analyzed in the range from 2 to 40,000 μg/kg in apple juice. Effects of heat pasteurization for patulin in apple juice Apple juice was usually pasteurized at 96 °C for 1 min. When the juice was pasteurized at 105 °C for 30 min or at 125 °C for 150 seconds, about 30 % of patulin was degradated (Table 1). However after these threatments the taste of the apple juice was severely damaged. So this type of physical treatment is not useful for apple juice.

Table 1. Effect of heat treatments on patulin in apple juice Treatment Patulin (%)

96 °C 1 min 100

105 °C 30 min 88

105 °C 60 min 69

120 °C 150 sec 73

125 °C 150 sec 70

130 °C 150 sec 68



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Figure 1. Chromatograms of patulin A: patulin standard (2 ng, 276 nm), B: apple extract (80 µg/kg, 276 nm) C: patulin standard (2ng, m/z=153), D: apple extract (80 µg/kg, m/z=153) Distribution of patulin in damaged apple and effect of trimming for its reduction A P. expansum was inoculted and cultured in ten Fuji apples. Each apple was divided into 36 pieces then correspond pieces (ten pieces for each position) were mixed and pressed. As shown in Figure 2, a large amount of patulin (1500 μg/kg) was detected from the portion that included the point at which the fungus was inoculated. Also, 930 μg/kg and 150 μg/kg of patulin were detected from the upper and lower side portion of the inoculation point and 8 to 20 μg/kg of patulin were detected from the other surrounding areas, including one section in the core area. However, no patulin was detected in the other 26 portions (16). This study was repeated three times using other apple cultivars and similar results were obtained each time. Because it is relatively easy to manually separate the area damaged by P. expansum from the visibly unaffected parts, the decayed parts of apples were carefully removed from the surrounding sound area. Two layers (ca. 5 mm thickness) of visibly unaffected parts of the damaged area were also taken. High concentrations of patulin (40 mg/kg, average of three samples) were detected in the juice extracted from the decayed portions. In the first layer extending up to 5 mm from the decayed section the concentration of patulin detected was 0.14 mg/kg (1:290 to compare to decayed area). In the layer taken 5-10 mm from the


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decayed area, the concentration of patulin detected was only 0.003 mg/kg (1:13000). These results clearly show that the distribution of patulin in apples that have decayed is mostly confined to the decayed areas. Therefore it should be possible to prevent patulin contamination of apple products made from decayed apples by the removal of the visibly decayed parts before processing. To confirm these results, the effects of trimming for patulin reduction were studied (16) and after trimming the decayed area with a knife, patulin was not detected from the remaining portion of apple.

Figure 2. Distribution of patulin in P. expansum inoculated apple.

Figure 3. Relation of decay size and concentration of patulin Each dot represents one apple. Four apple cultivars of apple and four strains of P. expansum were used.


75 Relation between size of decay and patulin accumulation on apple As shown in figure 3, there was a correlation between the decay size and the level of patulin. This result clearly showed that a small decay of less than 10 mm in diameter may not result in patulin contamination problem even if apples with such decay were to be used for processing without trimming.


Effect of storage temperature on P. expansum growth and patulin contamination on apple Four strains of P. expansum were inoculated onto apples and stored at 20, 5, 2.5, 1.0, -0.5 and -1 °C. Apples stored at less than -2.5 °C were frozen. As shown in Table 2, P. expansum can grow on apples at very low temperatures, even at -0.5 °C, but no patulin was detected in this sample even 83 days after inoculation. When apples were kept at 20 °C, decay started to be observed within two days after inoculation and the decay spread about 6 mm per day. The spread of decay on apples at 1 to 5 °C was 1 to 3 mm per day after decay was first observed. Interestingly, when apples were stored at low temperature, there was some lag time before decay started that depended on the storage temperature(17). For example at 5 °C, the lag time was 10 days to two weeks and at 1 °C, 2 to 4 weeks, depending on the strains of fungi and cultivar of apple. These results show that it is important to store apples in cool conditions as soon as possible after harvest to prevent patulin contamination. Table 2. Patulin production and decay caused by P. expansum Temp. (°C) 20 5 2.5 1 -0.5 -1 The day decay 2 14 21 35 60 60 was start to observed Stored period 8 60 60 63 83 63 (days) Decay (mm) after 61 70 55 23 4 2 storage*a Patulin 900 1300 350 300 N.D. N.D. (mg/kg)*a *a: Average of four P. expansum *b: Storage period is not applicable because frozen *c: Apple was frozen and no mold growth was observed *d: No analysis was done because apple was frozen N.D.: Not Detected (less than 0.5 µg/kg in the portion analyzed)


-2.5 *b *c *d

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Mycotoxin Prevention and Control in Agriculture - American Chemical

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