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Patulin in Fresh Fruits
The spoilage-causing mycotoxin presents its own unique challenges when it comes to detection procedures
by Christy Swoboda
Patulin, a natural food contaminant most often associated with fruits and fruit-based products, is a mycotoxin metabolite that obtained its name from the mold Penicillium patulinum. As a mutagenic, genotoxic, immunotoxic, and neurotoxic, patulin can be responsible for acute effects including nausea, vomiting, and other gastrointestinal issues. It can affect a developing fetus, the immune system, the nervous system, the gastrointestinal tract, and can potentially cause DNA damage.
It’s produced by several different fungi, but primarily from Penicillium species. Patulin has been detected in apples, pears, bananas, peaches, pineapple, blueberries, apricot, cherries, and grapes infected with Penicillium species. It has been associated recently with vegetables, cereal grains, and silage as it spoils. In a Food Standards Agency study which ran from 1998 to 2001, orchards were tested for the presence of Penicillium expansum. This fungi was found within the orchards in the soil, leaves, bark, fruit, and other orchard debris.
Patulin is associated with fruit, especially apples, exhibiting brown rot or other spoilage characteristics. Invading the fruit through insect damage, bruises, cracks, or other open spots, it can also affect varieties of fruit that exhibit an open calyx. It can be associated with poor storage resulting in spoilage of the fruit post-harvest, but prior to processing. There is an increased risk of toxin formation from long-term storage of raw fruits after harvest at ambient temperatures. Proper and safe harvesting of the fruit is critical to minimizing the risks for patulin formation. Steps to minimize damage to the fruit during harvest should occur. These steps can include the cleaning of harvesting bins prior to use on new crop, supervision to reduce bruising of the crop during harvest, and reduction of exposure of harvested fruit to adverse external environmental conditions. It is important to move harvested fruit to refrigerated storage within 18 hours of harvest—fruit that is left at ambient temperature for over a week to a few months shows a great increase in the risk of patulin formation from the field heat on those fruits.
Most often associated with apples, apple products, and apple juices within the U.S., the FDA believes that producers can control patulin by removing spoiled, bruised, moldy, and visibly rotting fruit prior to production as these fruits have an increased risk of toxin formation.
Patulin is very stable in fruit juices because the presence of sucrose within the juice actually protects patulin from degradation during heat treatments. A high risk of toxin formation is associated with fresh pressed juices or ciders made from spoiled or low quality fruits. Conversely, patulin contamination is often not associated with vinegars or alcoholic beverages due to interaction of the mycotoxin and yeasts during the fermentation process.
Testing for the presence of patulin in food products is not a simple or quick procedure. Current rapid test kits to detect the presence of patulin are lacking throughout the global market. The molecule of patulin is small in size and this has proven difficult for many antibody production companies to accommodate. In theory, it should be possible to produce such an antibody that could lead to a lateral flow device testing platform for a quick test of patulin presence in the production environment. Most patulin testing occurs via the use of HPLC (high performance liquid chromatography)-UV and/or liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS) analyses within a laboratory. Patulin does not employ fluorescent properties and thus the use of UV detection is required. Often times the chromatography for patulin analysis is complex. A compound known as HMF (5-hydroxymethylfurfural) often times co-elutes or presents close in retention time to the patulin peak of interest. Testing methods via HPLC-UV should include a HMF standard to confirm retention time and proper separation of this compound peak from the patulin peak for quantitation purposes to avoid the potential for false positives or elevated positive results.
Patulin is associated with fruit, especially apples, exhibiting brown rot or other spoilage characteristics.
As the popularity of LC/MS/MS methods for detection of mycotoxins continues to rise, more laboratories are turning to this technology to accurately detect patulin contamination at low levels of parts per billion (ppb). Couple this LC/MS/MS technology with the use of a C-13 Isotope Labeled Internal Standard for patulin and the method has great sensitivity and accuracy in even complex matrices. The proper use of the C13 Internal Standard allows the LC-MS/MS system to be equilibrated to matrix enhancements and matrix suppressions for accurate quantitation. Many laboratory methods can detect patulin contamination with limits of detection at 2 ppb.
Regulatory limits for the presence of patulin in food have been established in the European Union (EU) and China. To date official regulatory limits are not in place within the U.S.; however, recommendations or guidelines have been established for patulin inclusions within foods. For the U.S., these guidelines have been set at 50 microgram per kilogram (ug/kg) of patulin in apple juice, apple juice concentrates based upon single strength inclusion, and apple juice products. The guideline of 50 ug/kg by the FDA allows for a negligible risk of adverse health effects from the consumption of patulin within apple juice products over a routine period of time. China has established regulatory limits of 50 ug/kg in apple and hawthorn products. The EU has established regulatory limits of 10 ug/kg in apple juice and solid apple products for infants and young children, 25 ug/kg in solid apple products, such as apple puree, intended for direct consumption, and 50 ug/ kg in fruit juices and in drinks containing apple juice or derived from apples.
Proper sampling techniques must be employed in order to test for and detect the accurate presence of patulin within any raw fruit product or finished fruit puree, sauce, or juice. Based upon a study conducted by T.B. Whitaker and J.W. Dickens (1974), 98 percent of analytical error comes from improper sampling of the product being tested. Eighty-eight percent is attributed to sampling errors and 10 percent is attributed to sub-sampling errors from that initial sampling lot. Mycotoxins have a distribution problem within food and feeds—they are not evenly distributed across the lot of food being tested such as in proteins. Mycotoxins can exhibit clustering across the lot that can lead to either elevated or false positives above threshold or false negatives.
For proper sampling to occur, proper equipment must be utilized that pulls samples randomly from numerous spots across the lot. For raw fruits, this would include multiple samples from different bins or different locations of the bin. For purees, sauces, or juices, this would include multiple samples from different time points of the production process in order to capture a beginning, middle, and end sample at a minimum. The sampling plan should be an established protocol that is utilized in an identical pattern every time collection of a sample is to occur. By performing sampling in a routine and throughout random process, the confidence in an analytical result can be greatly improved.
The ultimate goal in sampling and analytical testing is to determine the accurate result. Ideally no patulin contamination will be detected within the fruit crops; however, should an occurrence of patulin be detected, it is critical for the sampling plan and analytical methodology employed to be sound and robust such that an accurate result is obtained for all involved parties.
Swoboda is laboratory director, quality manager at Romer Labs, Inc. Reach her at firstname.lastname@example.org.
References Furnished Upon Request