Preharvest

Although patulin production in fruit is believed to occur mainly postharvest, several factors pertaining to the growing conditions of fruit trees may influence fungal infection and mycotoxin production in apples. Codex Alimentarius Commission [92] outlined good agricultural practices (GAPs) that may reduce the likelihood of infection of fruit trees. Trees should be trimmed of dead and diseased wood and mummified fruits and pruned to allow proper air flow and light penetration [92]. It has been demonstrated that fruit with mineral imbalances are more susceptible to infection by P. expansum and other fungal pathogens. Supplementing fruit trees with foliar calcium sprays during the growing season and use of minimal amounts of nitrogen fertilizer are some methods for reducing preharvest infection of apple fruit by fungi [95,98]. Calcium is believed to reduce decay by maintaining the firmness of cell walls during ripening [98,113]. Ammonium molybdate tetrahydrate has been studied as foliar and soil treatment of several crops. When ammonium molybdate was applied as a preharvest treatment to apple trees, a significant reduction in blue mold decay was observed in the treated apples after three months' cold storage [114]. Tests in vitro showed that the mode of action of the chemical is by inhibiting germination of P. expansum spores [114].

Postharvest decay can be reduced by preharvest applications of fungicides. Studies on the effectiveness of applications of ziram fungicide showed an average reduction in decay of 25 to 50% [112,115]. Synthetic fungicides are being developed to protect produce from a number of postharvest diseases. However, problems associated with use of synthetic fungicides, such as proliferation of fungicide-resistant pathogen strains, as well as concerns about public health and environmental contamination, have increased the need for development of alternative treatments [116].

During the past five years, biological control of postharvest fungal diseases with naturally occurring antagonists (yeasts and bacteria) has become an alternative to synthetic fungicide control [116]. The commercial products Aspire™ (Candida oleophila strain 182, Ecogen Inc., Langhorne, PA) and Biosave™ 10 and BioSave™ 11 (Pseudomonas syringae strains ESC10 and ESC11, EcoScience Corp., Worcester, MA) are examples of commercial biocontrol products available in the U.S. Biocontrol agents act by colonizing the wounds of apples where decay proliferates. The organisms are believed to inhibit growth of fungal pathogens by utilizing all of the available nutrients in the wound. Although most success with biological control has been with application of the antagonists to fruit postharvest, but before storage, there has been some degree of success at preharvest treatment of apples with antagonists. Nunes et al. [117] reported that although preharvest treatments with Candida sake were less effective than postharvest treatments against P. expansion, about 54% control was achieved by spraying the organism on Golden Delicious apples while still on the tree. More work is needed to determine the efficacy of preharvest biocontrol of P. expansun and to determine if biocontrol affects postharvest formation of patulin in fruit.

13.8.3 Harvest

The condition of produce at harvest determines the length of time the crop can be stored [112]. Stage of maturity at harvest is believed to be one of the main factors determining the susceptibility of fruit to mechanical damage and to blue mold rot during postharvest storage. Fruits become increasingly susceptible to fungal invasion during ripening as the pH of the tissue increases, soluble sugars build up, skin layers soften, and defense barriers weaken [118,119]. To reduce undesirable biochemical changes, apples should be picked when mature but not fully ripe to ensure that they can be stored for several months [112].

Studies indicate that bruising and skin punctures substantially increase the susceptibility of fruit to decay. Gentle handling of fruit by pickers during harvest and care during transport of the fruit from the orchard to the packinghouse, juice processing plant, or storage may prevent injury to the fruit [73].

Rain during harvest allows for increased fungal contamination and infection [95]. Consequently, fruit should be harvested in dry weather conditions and quickly transferred to cold storage. Fallen fruit in the orchard should be discarded and not sold for the fresh market or used in processed apple products. Jackson et al. [87] reported significantly higher patulin levels in cider produced from ground-harvested apples than from tree-picked fruit. The process of falling from the tree may result in cuts or cracks in the apple peel that become infected from fungal spores from the soil.

One of the major methods for controlling P. expansun infection of apples is improved sanitary practices during harvest [95,120]. This includes reducing contamination of packing/storage bins with orchard soil by cleaning and sanitizing bins before use. Studies by Spotts and Cervantes [120] found that while steam was the most effective treatment on wood and plastic bins, chlorine compounds, sodium o-phenylphenate (SOPP), and quaternary ammonia compounds were also effective sanitizing agents. Sodium hypochlorite was more effective on P. expansun spores on plastic than on wood bins [120]. Benomyl, iprodione, and captan were generally not effective disinfectants.

13.8.4 Postharvest 13.8.4.1 Introduction

Approximately 75% of the world apple crop is marketed as fresh whole apples, with the remaining 25% finding its way into processing, primarily into apple juice and cider [121]. After harvest, a portion of the apple crop is transported to packinghouses where it is packaged for the fresh (table) fruit market.

Fruit not sold for the fresh market is processed into juice and other products, or is stored at cold (0 to 4°C) temperatures with or without modified atmosphere to extend the shelf life and to provide a constant supply of raw material for the fresh market and for the processed apple industry [122]. Since the majority of patulin forms in fruit postharvest, considerable efforts have been devoted to developing strategies for reducing proliferation of fungal pathogens and contamination with patulin during storage. This section outlines some of the major postharvest controls of patulin in apple products.

13.8.4.2 Washing Treatments

Organic matter (soil, plant material, decayed apples) can act as a reservoir of fungal spores that contaminate fruit. It is important to maintain sanitary conditions in all areas where fruit is packaged, stored, and processed. Proper sanitation includes washing and sanitizing packing machinery, the walls and floors of storage rooms, and the surfaces of all processing equipment [112].

Water systems (water flumes), used to float apples from field bins to bulk tanks, minimize mechanical damage to fruit. Flume water typically contains chlorine (sodium hypochlorite) or SOPP to reduce fungal spore load [112,123]. Active chlorine levels in flume water must be maintained periodically to ensure spores are destroyed. Other chemicals that can reduce spore levels include chlorine dioxide [124] and ozone [125], although both are not commonly used disinfectants for flume water. Physical removal of fungal spores by filtration has been reported to remove >92 to 99% of P. expansum conidia from flume water [126].

According to recent surveys of industry practices, the majority of apple packagers and processors wash apples upon receipt or immediately before chopping and pressing to remove soil, rot, pesticide residues, insects, microorganisms, and other extraneous material [127-129]. Apples are typically washed in dump tanks containing water or chlorinated water, with brusher-scrubbers, and/or with high-pressure water sprayers [122,128]. Since P. expansum and patulin are associated with the soft rot of apples, washing may result in the removal of rotten areas of the apple and the partitioning of patulin into the cleaning water [87,93]. Jackson et al. [87] found that washing ground-harvested apples in a dump tank before pressing reduced patulin levels in the resulting cider by 10 to 100%, depending on the initial patulin levels and type of wash solution (water vs. chlorine) used. Sydenham et al. [94] found that patulin levels in cider decreased from 920 to 190 mg/l after Granny Smith apples were washed with water. Acar et al. [130] reported that patulin levels were reduced by up to 54% when apples were washed with a high-pressure water spray. Total removal of patulin during the wash treatments is unlikely since patulin can diffuse up to 1 cm into healthy tissue [131]. Wash solutions other than chlorine that have had efficacy in reducing mold counts in apples include electrolyzed oxidizing water [132] and ozone [125], although their effects on the patulin content of apples are not known.

13.8.4.3 Culling, Sorting, and Trimming

Removal of decayed or damaged fruit or trimming moldy portions of apples prior to packaging or processing have been reported to reduce patulin levels in apple juice [4,93,94,131,133]. Wilson and Nuovo [76] surveyed 100 samples of fresh cider and found that samples having the highest patulin levels were produced by cider mills that did not remove decayed apples before pressing. Similarly, Sydenham et al. [93,94] reported that removal of rotten fruit prior to pressing significantly reduced patulin levels in cider produced from apples stored at ambient temperatures for 7 to 35 days. Jackson et al. [87] reported that patulin was not detected when apples were culled prior to pressing, but was found in five out of seven varieties when cider was pressed from unculled fruit. Although removing visibly decayed fruit before processing is a proven method for reducing patulin levels in apple products, there is no guarantee that culling alone can totally eliminate patulin. Apples with "invisible" sources of fungal rot (core rot) can contaminate apple juice, cider, or puree with patulin if they are not removed before processing. Apple cultivars susceptible to core rot should be cut in half and fruit with signs of decay removed before processing. In large-scale operations where this culling procedure is not practical, other methods for detecting apples with core rot are needed.

13.8.4.4 Chemical, Heat, and Biological Control, and Irradiation Treatments

Prior to storage, apples are often drenched with diphenylamine along with a fungicide (thiabendazole) to prevent superficial scald [104]. Since some strains of pathogens are developing resistance to fungicides, there has been a push to use alternative postharvest control methods [113,134]. Treatments that have shown some promise include the use of essential oils [135], organic acid fumi-gants [136], calcium salts [98], carbonate and bicarbonate [137,138], chitosan [139-141], 2-deoxy-D-glucose [142,143], heat, biological control, irradiation [118], and combinations of these treatments.

Spraying cinnamon oil, cinnamaldehyde, or a potassium sorbate solution on the surface of apples extended shelf life with respect to decay by P. expansum [135]. Complete inhibition of patulin formation in liquid culture was found with 0.2% lemon oil, and >90% inhibition was observed using 0.05% lemon oil and 0.2% orange oil [60]. It is unclear if these treatments can be used commercially to reduce fungal decay in apples or if they affect the shelf life of fruit.

Preliminary studies by Sholberg et al. [136] indicate that fumigation of fruit with short-chain organic acids prevents decay and could become an important alternative to liquid sterilants such as sodium hypochlorite. The number of lesions on apples caused by P. expansum decreased exponentially with increasing time of fumigation with vinegar or acetic acid vapors [136,144]. Drawbacks to the use of acid fumigants include the need for an airtight enclosure and the corrosiveness of the vapors to steel [136].

Fungal decay in apples was reduced by postharvest application of calcium solutions to fruit [98,113]. Direct application of calcium to fruit can be accomplished by dipping or spraying fruit with calcium solutions or with vacuum or pressure infiltration [99]. Calcium helps to maintain firmness of the apple and to decrease the incidence of physiological disorders that enable fungal pathogens to infiltrate the fruit tissue.

There has been an increased interest in the use of prestorage heat treatments to prevent fungal decay of fruit. Heat can be applied to fruit as a hot water dip, as steam, as hot dry air, and by short hot water rinses [145-148]. Leverentz et al. [134] reported that holding Golden Delicious apples at 38°C for four days reduced decay after three months of storage at 0°C without reducing fruit quality. Fallik et al. [148] reported that Golden Delicious apples treated with a 15-second hot water (55°C) rinse followed by a brushing treatment had less P. expansum decay than untreated apples or apples given a dry heat treatment (96 hours at 38°C). One explanation for the enhanced stability of the heat-treated fruit is that heated apples softened more slowly than nonheated fruit. In addition, heat treatments may recrystallize the wax layer on the surface of the apple peel or increase synthesis of wax in the peel [148,149].

A promising alternative to chemical treatments is biological control of postharvest pathogens [116,117,134,150,151]. Decay caused by P. expansum has been controlled in pome fruits by bacterial and yeast antagonists in several laboratory and pilot storage tests [134]. At least one yeast-based product and two bacteria-based products are now commercially available for treating apples after harvest. Several more are being developed for commercialization [151-153]. Although biological control agents have exhibited excellent control of fungal rot in fruit, their efficiency is sometimes lower than chemical control, and they do not always give consistent results [112,153]. Microbial antagonists have a poor ability to eradicate preexisting infections, while chemical treatments are frequently more effective at controlling established infections [153]. Use of a combination of microorganisms could improve the spectrum of activity and reduce the required concentration of biocontrol agents [116]. El Ghaouth et al. [139,140] reported enhancing the biological efficacy of the yeast Candida saitoana by combining it with either glycochitosan or with the sugar 2-deoxy-D-glucose. Both approaches increased the protective and curative activity of the yeast in controlling postharvest diseases. Droby et al. [153] found that application of 2% sodium bicarbonate in combination with AspireTM consistently enhanced its biocontrol performance against penicillium rot in apples. Similarly, McLaughlin et al. [154] demonstrated that the addition of calcium salts to yeast cell suspensions enhanced the ability of Pichia guilliermondii to control postharvest diseases of apple. Pichia guilliermondii is found as an occasional clinical isolate and therefore is of questionable safety as a biocontrol organism [155]. In another study, a combination of a heat treatment and the use of a yeast antagonist was more effective than either treatment alone [134]. Clearly more work is needed to identify a combination of treatments to control penicillium rots in apples. Additional research is also needed to determine if these treatments are able to inhibit patulin formation in fruit [153].

Aziz and Moussa [118] studied the effect of gamma irradiation on mycotoxin production in fruits stored under refrigeration conditions. After 28 days of storage, nonirradiated fruits were contaminated with higher levels of mycotoxins (including patulin) than irradiated (3.5 kGy) samples. Mycotoxin production was reported to decrease with increasing irradiation dose. Although UV light has a lethal effect on bacteria and fungi, little has been done to study the effects of UV irradiation on mold levels on apples. However, Stevens et al. [156] reported that applying a yeast antagonist to fruit after UV irradiation was the most effective treatment in reducing storage rot in peaches. Use of gamma and UV irradiation to control fungal rot in fruit deserves further study.

13.8.4.5 Storage

After harvest, apples are generally kept in cold storage at —1 to 3°C with or without modified atmospheres. These treatments can extend the shelf life of apples from 9 to 28 weeks, depending on apple cultivar [109]. Since apple cultivars differ in their susceptibility to postharvest diseases, cultivars with resistance to mechanical damage and infection should be chosen, especially if they will be kept in long-term storage.

Although fungal growth is dramatically reduced at temperatures < 10°C, the growth of P. expansum and production of toxin were not prevented during cold storage [89,90,157]. Paster et al. [90] found that patulin levels in apples and pears inoculated with different strains of P. expansum generally increased with increasing storage temperature from 0 to 25° C. Similarly, Beer and Amand [89] reported that Macintosh apples stored at 4°C had substantially lower patulin levels than fruit stored at 15 or 24° C. Apples are typically kept in cold storage; however, when suitable refrigerated storage is not available, apples are stored in the open in ambient conditions (i.e. deck storage). Sydenham et al. [94] reported that patulin levels in deck-stored apples were 2445 mg/l as opposed to 90 mg/l in comparable refrigerated stored fruit. Fungal growth and patulin formation increased with the length of storage [90,94]. Overall, the research presented here indicates that apples should be kept in refrigerated storage when possible to slow mold growth and reduce mycotoxin production.

Several researchers studied the effects of modified atmosphere conditions and found that gas composition affected P. expansum growth and patulin formation in fruit [90,158-160]. Lovett et al. [71] reported that juice from modified atmosphere-stored apples (3% O2, 1 to 3% CO2, 0 to 3.3°C, and >90% relative humidity; 14 weeks) had 500 mg patulin/l while juice made from air-stored apples had 2000 to 3000 mg patulin/l. Stitton and Patterson [160] reported that use of high (>3%) CO2 atmospheres (greater than used for commercially stored apples) was an effective fungistatic treatment for stored apples. However, excessively high levels of CO2 (>8%) negatively affected the quality of some apple cultivars. Johnson et al. [159] reported a lower incidence of penicillium rots in apples stored at lower O2 conditions (0.75% O2) than under higher O2 levels (1.0 to 1.25%). While in modified atmosphere storage, apples should be examined periodically for fungal decay [92].

13.8.4.6 Controls for Processed Apple Products

Treatments that have shown promise at reducing patulin levels in apple juice include filtration, centrifugation, use of charcoal, addition of ascorbic acid, and fermentation [16,109,161-164]. Bissessur et al. [164] evaluated the effectiveness of several clarification processes for the reduction of patulin in apple juice. Pressing followed by centrifugation resulted in 89% reduction in levels of the toxin. Patulin reductions using paper filtration, enzyme treatment, and fining with bentonite were 70, 73, and 77%, respectively. These data suggest that patulin tends to bind to the apple solids, which are removed from the juice during treatment. Activated carbon treatment has also shown promise as a method for reducing patulin levels in apple juice [163,165,166].

Several compounds have the ability to modify chemically patulin, rendering the toxin undetectable in some analyses. Yazici and Velioglu [167] found that adding vitamins (thiamine hydrochloride, pyridoxine hydrochlor-ide, and calcium-d-pantothentate) to apple juice before storage at 4°C for 6 months reduced patulin levels by 55.5 to 67.7% versus controls (no vitamin addition) that had 35.8% reduction in levels of the toxin. It is unlikely that the use of these vitamins to reduce patulin levels in juice has any practical value. Adding ascorbic acid (0 to 3% w/v) to apple juice has been reported to reduce patulin levels by up to 80%, as measured by HPLC [16]. The mechanism by which patulin interacts with ascorbic acid needs to be studied in more detail.

Patulin is known to become analytically undetectable during the production of cider from contaminated apple juice [18,168]. Analysis of patulin-spiked fermentations by HPLC showed the appearance of two major metabolites of patulin, one of which appeared to be E-ascladiol [18]. More work is needed to determine the toxicity of these metabolites of patulin.

Cure Your Yeast Infection For Good

Cure Your Yeast Infection For Good

The term vaginitis is one that is applied to any inflammation or infection of the vagina, and there are many different conditions that are categorized together under this ‘broad’ heading, including bacterial vaginosis, trichomoniasis and non-infectious vaginitis.

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