Spoilage Of Fresh Mushrooms

Quality is the single most important factor affecting retail mushroom sales [21]. Whiteness, cleanliness, and brown blotches on fresh mushrooms are the principal factors determining mushroom quality. Consumers prefer to purchase mushrooms that are bright white, free of casing material or other unwanted particulate contaminants clinging to the mushroom surface, and free of brown blotches. The brown blotch discoloration of mushrooms is perceived as a symptom of decreased freshness or microbiological deterioration (spoilage).

Enzymatic browning catalyzed by the enzyme tyrosinase (polyphenol oxidase) [22] is the most important factor involved in quality deterioration of fresh mushrooms. The browning reactions are initiated by tissue breakdown due to either mechanical damage or bacterial activity [23]. It has been suggested that the role of tyrosinase in mushrooms is to function as a stress metabolite [24]. Tyrosinase naturally occurs at high levels in the mushroom surface tissue, and is normally found in a latent form [5]. When activated during senescence [22] the enzyme oxidizes mushroom phenolic compounds into brown melanins [25-27] resulting in brown discoloration. In fresh mushrooms, tyrosinase and its substrates have been hypothesized to be located in separate subcellular compartments [22]. When mushrooms are mishandled or bruised the cellular membrane is damaged, and rapid browning of the mushroom cap is observed. It has been hypothesized that the loss of membrane integrity provides greater access of tyrosinase to its substrates, resulting in formation of brown compounds [22,28] and associated brown discoloration of fresh mushrooms.

The presence of high bacterial populations in fresh mushrooms is a major factor that significantly diminishes quality by causing a brown, blotchy appearance [23] (Figure 6.3). The rate of postharvest deterioration of fresh mushrooms has been directly related to the initial microbial load [23]. Doores et al. [19] found that bacterial populations during postharvest storage at 13°C increased from an initial load of 7 log CFU/g to almost 11 log CFU/g over a 10-day storage period. The authors also reported that deterioration of mushroom quality as indicated by maturity and color measurement appeared to be concomitant with increase in bacterial numbers. Pseudomonas spp. and Flavobacterium spp. were the two main groups that predominated during agaricus mushroom postharvest storage. Similarly we have observed that bacterial populations tend to increase from 7.3 to 8.4 log CFU/g during a 6-day storage period at 4°C (Figure 6.4). Populations of yeast increased from 6.9 to 8.0 log CFU/g during the storage period. Populations of molds remained constant (3 log CFU/g) during the storage period [29,30].

A majority of mushrooms of good quality and color, harvested and marketed, develop blotches at retail or in consumer homes, even while kept at refrigeration temperatures. Symptoms of brown blotch disease are sunken, dark, and brown spots [31] on the mushroom fruit body surface. Pseudomonas is the major spoilage genus associated with blotch

FIGURE 6.3 Scanning electron micrographs of mushroom cap surfaces: (A) healthy tissue (x3000); and blotched tissue showing invading bacteria (B) (x3000) and (C) (x 10,000).

FIGURE 6.4 Increase in aerobic bacterial populations and a concomitant decrease in the whiteness (measured by L-value) of fresh Agaricus bisporus mushrooms during postharvest storage at 12°C. The solid line represents aerobic bacterial populations (log CFU/g fresh mushroom tissue). The broken line represents the L-value of the mushroom cap during postharvest storage. Data are the average of four independent samplings.

■ Bacterial population (Log CFU)

FIGURE 6.4 Increase in aerobic bacterial populations and a concomitant decrease in the whiteness (measured by L-value) of fresh Agaricus bisporus mushrooms during postharvest storage at 12°C. The solid line represents aerobic bacterial populations (log CFU/g fresh mushroom tissue). The broken line represents the L-value of the mushroom cap during postharvest storage. Data are the average of four independent samplings.

formation of fresh mushrooms [32-34]. Paine [35] attributed Pseudomonas tolaasii as the causative organism of the classic bacterial blotch disease of cultivated mushrooms. Application of P. tolaasii cells as low as 20 CFU/cm2 of growing beds resulted in blotch formation in mushrooms [36]. Symptoms of mushroom blotch became visible when 5.4 106 CFU/cm2 were detectable in the mushrooms [36]. When P. tolaasii was placed directly onto caps, 6 x 107 CFU/cm2 were necessary to produce a blotch lesion (though only 3.5 x 106CFU could be recovered). The researchers of the study [36] concluded that the number of cells of P. tolaasii present in the early primordial stages of mushroom growth controls the extent of blotch disease seen at harvesting. It has also been shown that tyrosinase is activated during infection by the bacterium P. tolaasii or exposure to its toxin, tolaasin, causing brown blotch disease symptoms of fresh mushrooms [37]. Wells et al. [38], by isolating and reinoculating the bacteria on freshly harvested healthy mushrooms, confirmed that postharvest blotch formation and associated discoloration was caused by three phenotypic groups (pathotypes) of fluorescent pseudomonads. Severe infections with darkened or yellowed lesions were caused by strains of pathotypes A or B, respectively. Mild infections with superficial discoloration were caused by the pathotype C. Based on cellular fatty acid analysis, the authors concluded that each pathotype corresponded to one or several mushroom-related pseudomonads reported in the literature as follows: pathotype A = Pseudomonas tolaasii, pathotype B = Pseudomonas "gingeri", and pathotype C = Pseudomonas ''reactans''. Isolates from mushroom casing material yielded all three pathotypes.

Fluorescent pseudomonads also produce exopolysaccharides (EPSs) associated with the sliminess accompanying spoilage of mushrooms. Fett et al. [39] isolated, partially purified, and characterized acidic EPSs from 63 strains of mushroom-associated fluorescent pseudomonads. The strains were originally isolated from discolored lesions on mushroom caps, or from commercial lots of mushroom casing soil. An acidic galacto-glucan named marginalan was produced by mucoid strains of the saprophyte Pseudomonas putida and the majority of mucoid strains of saprophytic P. fluorescens isolated from casing medium. Other strains produced EPSs that included alginate, and unique EPSs containing neutral and amino sugars and glucuronic acid.

There has been a long and complex association between the fungal genus trichoderma and mushroom cultivation since Beach [40] first reported disease symptoms on caps of agaricus mushrooms. In a study by Sharma et al. [41] colonization assessments confirmed that Trichoderma harzianum biotypes Th1, Th2a, Th2b, and Th3 inoculated into the mushroom substrate became established in the mushroom substrate. The extension rate of two Th2 isolates in the substrate was over 1000 times that of Th1 and Th3. Results confirmed that while Th1 and Th3 did not significantly affect yield, Th2 could reduce mushroom quality and productivity by as much as 80%. In vitro studies by Mumpuni et al. [42] suggested that the growth of T. harzianum biotypes could be related to the release of metabolites by A. bisporus into the compost substrate. Dilute aqueous solutions of n-butanol extracts of A. bisporus culture filtrates and fruit bodies inhibited Th1 and Th3 but stimulated Th2 isolates, suggesting that the active compound(s) may be constitutive components of the A. bisporus species.

6.3.1 Sources of Microorganisms Causing Spoilage

It has been demonstrated that the casing microflora have a vital role in the sporophore (fruit body) formation of mushrooms from the mycelia stage [43-45]. The requirement for biotic agents in the initiation of fruit body formation [45] excludes the possibility of mushroom cultivation on a commercial scale under axenic conditions. This factor, combined with the intensity of production within a confined area, results in the introduction of microorganisms on fresh mushrooms that contribute to spoilage during postharvest storage.

The casing layer on which the mushroom fruiting bodies develop is a significant reservoir for the microflora of fresh mushrooms [19]. Doores et al. [19] found that aerobic bacterial populations from casing material ranged between 8.2 and 8.5 log CFU/g. In a study conducted by Wong and Preece [46], the primary sources of Pseudomonas tolaasii on a mushroom farm were the peat and limestone used in the casing process. This mushroom pathogen could not be detected in the farm soil, water supply, the mushroom spawn used, or in compost after spawning, but was isolated from the casing (peat/limestone mixture) layer of symptom-free mushroom beds and both the casing layer and compost of beds bearing blotched mushrooms. Secondary sources were numerous once the pathogen was present in mushroom beds. These included symptomless and blotched mushrooms, the fingers and shoes of people handling the crop, their baskets, knives, and ladders. P. tolaasii was also isolated from dust in the air of infected houses. While spores of infected mushrooms may transport the bacterium, sciarid flies can act as vectors contributing to bacterial transfer.

6.3.2 Cultural (Growing) Practices Favoring Spoilage

The extent of irrigation significantly affects the bacterial populations and the quality of the mushroom crop. Wong and Preece [36] concluded that very frequent irrigation, resulting in over-watering, increased blotch symptoms on mushrooms during growing.

6.3.3 Cultural Practices to Suppress Spoilage of Fresh Mushrooms

Significant efforts have been directed to improve mushroom quality by adding calcium salts or antimicrobial treatments to irrigation water during cultivation. Barden et al. [47] demonstrated that the postharvest shelf life of fresh mushrooms increased by 2 days when mushrooms were irrigated with 0.5% calcium chloride. The increase in shelf life was mainly due to a decreased rate of postharvest bacterial growth and a concomitant reduction of surface browning. Solomon et al. [48] demonstrated a significant improvement in quality and shelf life when mushroom crops were irrigated with tap water containing 50ppm stabilized chlorine dioxide and 0.25% calcium chloride. Initial and postharvest bacterial counts and degree of browning were lower in these mushrooms as compared to mushrooms irrigated with water without chlorine dioxide or calcium chloride. Irrigation treatments involving the addition of calcium salts to irrigation water to reduce bacterial populations and improve initial and postharvest mushroom quality have been extensively studied [10,24,48,49], and are now a common commercial growing practice.

Kukura et al. [11] conducted a study to examine the influence of 0.3% CaCl2 added to irrigation water on mushroom tyrosinase activity and postharvest browning. With the addition of CaCl2 to the irrigation water, the calcium content of mushrooms significantly increased, accompanied by reduced postharvest browning. Irrigation with CaCl2 had no effect on inherent tyrosinase activity. The CaCl2 irrigation treatment had even more pronounced improvement on mushroom shelf life following a standard bruising treatment, as indicated by reduced browning. Based on transmission electron micrographs, the authors speculated that increased levels of calcium in mushrooms irrigated with CaCl2 may have decreased browning by increasing vacuolar membrane integrity, thereby reducing the opportunity for tyrosinase to react with its phenolic substrates.

In other studies in our laboratory [29] we evaluated irrigation with modified acidic electrolyzed oxidizing (EO) water in combination with 0.3% calcium chloride on the reduction in bacterial populations of fresh mushrooms. Crops were grown using standard growing practices except for the experimental additions to the irrigation water of acidic EO water (diluted with 2 parts of regular irrigation water) and/or 0.3% calcium chloride. Compared to the control, all treatments reduced bacterial populations on the fresh mushrooms. While no significant differences in color were observed between the treatments on the day of harvest, irrigation with modified acidic EO water and/ or calcium chloride resulted in enhanced whiteness, point-of-sale appearance, and quality after a 7-day holding period of the fresh mushrooms. Recently we investigated the effect of irrigation with water containing 0.75% hydrogen peroxide on reduction in bacterial populations on fresh mushrooms. Irrigation with 0.75% hydrogen peroxide in combination with 0.3% calcium chloride added to the irrigation water consistently reduced the bacterial populations on fresh mushrooms by 85% (compared to bacterial populations on mushrooms irrigated with water without hydrogen peroxide and calcium chloride). This irrigation combination treatment shows promise as an effective preharvest method to enhance the quality of fresh mushrooms.

Research has been conducted to investigate the effect of natural antimicrobial secondary metabolites added into the irrigation water. In a study by Geels [50], a 1% aqueous solution of kasugamycin, an antibiotic produced by Streptomyces kasugaensis, was evaluated for reducing bacterial blotch after artificial infection of the mushroom crop with P. tolaasii. An artificial infection was established in the first flush (harvest) by inoculating the button-sized mushrooms with a suspension of P. tolaasii. A 1% aqueous solution of kasugamycin supplied through irrigation water on the second-flush mushrooms drastically reduced bacterial blotch symptoms on these mushrooms at picking stage. Disease incidence in the second flush in the control treatment (inoculated with P. tolaasii) was composed of 18% lightly, 29% moderately, and 10% heavily affected mushrooms, which totaled to 57% affected. The 1% kasugamycin treatment significantly reduced total disease incidence to only 9% (lightly) affected. In the same study, a sodium hypochlorite-based irrigation treatment showed no beneficial results.

Studies with canned products processed from mushrooms grown under experimental cultural conditions indicated that canned product spoilage was reduced significantly by employing peat versus soils as the casing material [51]. While this study has no implication on the spoilage of fresh mushrooms, it does indicate that casing type may have an effect on the microbiology and microbial spoilage of fresh mushrooms.

Aerated steam treatment is sometimes employed to treat thermally (pasteurize) the casing layer. Though steam treatment of casing material is not a common cultural practice, some commercial growers employ pasteurization (60° C, 140° F) of the casing layer to control diseases associated with some materials they employ. However, most growers do not heat-treat their casing material because of the additional cost involved and anecdotal evidence that crop yield will be reduced.

It has been speculated that reducing the microbial load in the casing layer may result in reduced bacterial populations associated with the mushrooms and improve postharvest quality [23]. Hence, we conducted an experiment to evaluate casing pasteurization on reduction in bacterial populations in fresh mushrooms and its effect on crop yield and quality. The crop was grown at the Mushroom Test Demonstration Facility (MTDF) on the Penn State University campus using standard growing practices used at the MTDF except for the pasteurization treatment to the mushroom casing. Unpasteurized casing served as the control. For pasteurization, the casing material was held in a steam vault designed for direct steam injection. Steam was generated on-site. Pasteurization of the casing was conducted by forcing a mixture of air and steam into the vault to increase the temperature of the casing material to 60°C (140°F). The casing material was held at 60°C for at least 2 hours. Following the application of the pasteurized and untreated (control) casing layers to the colonized compost the rest of the growing, irrigation, and harvesting procedures were conducted as per standard MTDF practices. Pasteurization of the casing layer (Figure 6.5) resulted in a 2.9 log CFU/g reduction in total bacterial populations (reducing the total population in the pasteurized casing from 5.9 to 3 log CFU per gram of casing layer material). However, bacterial numbers of the pasteurized casing

ISSS Unpasteurized casing I I Pasteurized casing

Pasteurization/ pre-irrigation

Pasteurization/ 1-week irrigation

FIGURE 6.5 Effect of pasteurization at 60°C followed by irrigation on total bacterial populations in the mushroom casing soil.

Pasteurization/ pre-irrigation

Pasteurization/ 1-week irrigation

ISSS Unpasteurized casing I I Pasteurized casing

FIGURE 6.5 Effect of pasteurization at 60°C followed by irrigation on total bacterial populations in the mushroom casing soil.

Unpasteurized casing

I I Pasteurized casing

Casing treatments

FIGURE 6.6 Aerobic bacterial populations on fresh Agaricus bisporus mushrooms grown using either pasteurized or unpasteurized casing soil.

Casing treatments

Unpasteurized casing

I I Pasteurized casing

FIGURE 6.6 Aerobic bacterial populations on fresh Agaricus bisporus mushrooms grown using either pasteurized or unpasteurized casing soil.

increased by 3.9 log CFU/g (from 3 log to 6.9 log) following 1 week of irrigation. At the same time the bacterial numbers increased by 1.4 log CFU/g in the unpasteurized casing (from 5.9 log to 7.3 log). Interestingly, there was no significant difference in bacterial numbers in mushrooms grown using unpasteurized or pasteurized casing (Figure 6.6). Mushrooms grown on steam-treated casing material showed improved postharvest shelf life. However the crop yield decreased by 10% when the pasteurized casing was used.

From a food safety perspective, a recommendation to steam-treat mushroom casing soils to reduce pathogenic bacterial populations should be delayed, since steam treatment may negatively affect a hurdle (beneficial soil microflora) to inhibit foodborne pathogens introduced into the soil (via irrigation water or cross contamination). Preliminary research in our laboratory has indicated that survival of Listeria monocytogenes is enhanced in pasteurized casing soil (60°C, 2 hours), compared to untreated soil. Under mushroom growing casing conditions (80% moisture, 22°C), 6.8 log CFU/g of L. monocytogenes was reduced to undetectable levels in 10 days in untreated casing soil. During this time period, populations of L. monocytogenes remained unchanged in pasteurized casing soil. So far, we have been able preliminarily to identify that the Penicillium sp. present naturally in casing soils may play a vital role in the destruction of L. monocytogenes. It is possible that thermal pasteurization of casing soil may destroy the penicillium and other beneficial microbial populations, thereby allowing survival of L. monocytogenes in the casing soil. Hence practical nonthermal methods are urgently required to destroy selectively the foodborne pathogens without significantly affecting the beneficial microbial populations in casing soils. Interestingly, L. monocytogenes demonstrated enhanced survival in casing soils colonized with the agaricus mycelia than in soils without the mycelia present in it. This situation warrants research on casing soil handling and disinfecting crop irrigation procedures to achieve preharvest food safety and quality goals.

Biocontrol has been evaluated as an alternative cultural practice to reduce bacterial populations and subsequently enhance quality and postharvest shelf life. Nair and Fahy [52] reported the isolation of three bacteria antagonistic to P. tolaasii from soil and peat. These were a nonfluorescent Pseudomonas species from soil, and strains of P. fluorescens and Enterobacter aerogenes from peat. When the antagonists and the pathogen (Pseudomonas tolaasii) were added in the ratio of 7.9:6 log CFU/ml to unsterilized peat and applied to mushroom trays, infection of mushroom sporophores by the pathogen was effectively controlled. In vitro studies failed to show lysis or growth inhibition of P. tolaasii by the antagonists. While biocontrol-based products have been introduced into the market in the recent past for controlling bacterial brown blotch of mushrooms, they have not been a significant commercial success.

6.3.4 Postharvest Conditions Favoring Spoilage of Fresh Mushrooms

Postharvest storage conditions significantly contribute to mushroom quality and shelf life. Pai [53] evaluated the effect of storage temperature (5, 10, and 15°C), and relative humidity (RH) (91, 94, 97, and 99%) on weight loss, whiteness change, and microbial activity of A. bisporus mushrooms. Weight loss of tested samples was correlated highly with storage time at each RH level. Increasing storage temperature and decreasing RH significantly enhanced (p < 0.05) the rate of weight loss. Mushroom whiteness values were not affected (p > 0.05) by changes in RH. Microbial growth increased with increasing storage temperatures. It was concluded that the use of clean mushrooms with low initial microbial counts, an environment of high RH, and minimal condensation in packages are important factors for maximizing the shelf life of mushrooms under refrigerated storage.

Temperature abuse during storage is an important factor contributing to the spoilage of fresh mushrooms. Tano et al. [54] evaluated the effects of temperature fluctuation on the atmosphere inside modified atmosphere containers and their impact on the quality of fresh mushrooms within the containers. Mushrooms were packaged in 4-liter modified atmosphere (MA) containers, and an atmosphere of 5% O2 and 10% CO2 was maintained at 4°C. Temperature was fluctuated from 4 to 20°C during a 12-day storage period in cycles of 2 days at 4°C followed by 2 days at 20° C. The severity of bacterial blotch on mushrooms was assessed using a rating of 1 to 4, with 1 = no bacterial blotch and 4 = above 25% of the mushrooms cap area with symptoms of blotch disease. Temperature increase during fluctuations caused anoxic atmospheres both in O2 (1.5%) and CO2 (22 to 10%). The quality of mushrooms stored under temperature fluctuating regime was severely affected as indicated by extensive browning, loss of firmness, and a high level of ethanol in the tissue compared to mushrooms stored at constant temperature. For the control group, the bacterial blotch index was negligible over a 6-day storage period, whereas with mushrooms stored under temperature abuse conditions, the index increased rapidly from 2.6 to 3.6 after 4 days. This study clearly demonstrated that temperature abuse and temperature fluctuation seriously compromise the benefits of MA packaging of fresh mushrooms.

Condensation of water in packages can severely affect the quality of fresh packaged mushrooms. Apart from making the appearance of the mushroom packs unattractive, condensation is not desirable since a water layer on mushroom caps supports the growth of Pseudomonas tolaasii [55]. Gormley and MacCanna [56] studied the effect of overwrapping mushrooms with different types of perforated and nonperforated films on changes in mushroom quality during storage. They found that water condensation occurred on the underside of the nonperforated film. At the same time excessive water loss through the perforated films caused wrinkling and brown patches on the mushroom caps [56]. Hence it is important to select packaging material taking into consideration the high respiration rate of mushrooms and the potential fluctuating storage temperatures during warehouse storage and retail display.

6.3.5 POSTHARVEST PRACTICES TO SUPPRESS SPOILAGE of Fresh Mushrooms

Various postharvest treatments have been investigated in order to impede browning and reduce rate of spoilage of fresh mushrooms. While proper cold storage is a primary requirement during postharvest storage, new or novel packaging techniques, washing treatments, and irradiation of mushrooms can further contribute to spoilage suppression [2].

6.3.5.1 Packaging

Overwrapping mushrooms with plastic film improves their quality as observed by rate of cap opening, color, and weight loss [56-58]. Since mushrooms respire heavily (500 mg CO2/kg fresh weight/hour at ambient temperature) [59], it is important to ensure proper ventilation of the packages to maintain a high O2 environment within the packages. Freshly harvested mushrooms were found to induce a near anaerobic environment (<2% O2) in unventilated, PVC-overwrapped packages within 2 to 6 hours when incubated at 20 to 30°C [60]. To prevent in-package atmospheres from turning anaerobic which can increase risk of Clostridium botulinum growth, conventional mushroom packages are also perforated at the top with 2 mm holes in accordance with a U.S. Food and Drug Administration (FDA) recommendation [61].

New technologies such as modified atmosphere packaging (MAP) have been developed in order to delay quality loss and to extend storage life of mushrooms [62-64]. The MAP method changes the mixture of gases surrounding a respiring product to a composition other than that of air. The gas composition of a storage atmosphere may reduce both microbial and physiological spoilage of fresh mushrooms [65] Lopez-Briones et al. [66] demonstrated that while up to 2.5% CO2 seems to benefit mushroom whiteness, CO2 concentrations higher than 5% enhanced mushroom discoloration during storage. The authors suggested that a desirable modified atmosphere for mushrooms storage should contain 2.5 to 5.0% CO2 and 5 to 10% O2.

Water persisting on mushroom caps after irrigation supports the growth of Pseudomonas tolaasii [55] and subsequent appearance of blotch. Roy et al. [67,68] evaluated sorbitol as a moisture absorber in mushroom packages at 12°C. Surface moisture content of mushrooms decreased in the presence of a sorbitol pouch. Mushrooms packaged with 10 g sorbitol pouches had constant surface moisture content and those packaged with 15 g sorbitol pouches had the best overall color. Lowering the in-package relative humidity did not affect the maturation rate of mushrooms but reduced bacterial growth, suggesting that improvement in color was probably due to reduced bacterial activity.

Martin and Beelman [60] evaluated the potential of Staphylococcus aureus to grow and produce staphylococcal enterotoxin in ventilated and unventilated fresh mushroom packages when stored at 25 to 35°C. Mushrooms were inoculated with an enterotoxigenic strain of S. aureus and incubated in overwrapped trays at different temperatures. S. aureus grew and produced staphylococcal enterotoxin (SE) in unventilated PVC-overwrapped mushroom packages when inoculated at levels of 3, 4, and 5 log CFU/g of mushroom after 4 days of incubation at 30° C. Growth of S. aureus was observed at all levels of inoculation at 25°C, but no SE was detected after 7 days of incubation. When mushroom packages were ventilated, S. aureus growth was suppressed and no SE was detected after 7 days at 25°C and 4 days at 30° C. However, S. aureus growth in ventilated packs exceeded growth in unventilated packages when the incubation temperature was increased to 35° C; SE was detected within 18 hours of incubation at this temperature, even in mushrooms inoculated at a low level (2 log CFU/g). These results show the extreme importance of proper sanitation and worker hygiene during mushroom harvesting and packaging, ventilation of fresh mushroom packages, and use of proper storage temperatures for fresh mushrooms at all points of the food chain since SE is extremely thermotolerant and can even survive the rigorous thermal process used in canning mushrooms [69].

6.3.5.2 Washing Treatments

Washing mushrooms has recently gained commercial popularity as a means of removing casing soil particles and for the application of browning and microbial inhibitors. Prior to 1986, aqueous solutions of sulfite, particularly sodium metabisulfite, were used to wash mushrooms for the purpose of removing unwanted particulate matter and to enhance mushroom whiteness. While sulfite treatment yielded mushrooms of excellent initial whiteness and overall quality, it did not inhibit the growth of spoilage bacteria. Therefore, the quality improvement brought about by sulfite use was transitory. After 3 days of refrigerated storage, bacterial decay of sulfited mushrooms becomes evident. In 1986 the FDA banned the application of sulfite compounds to fresh mushrooms due to severe allergic reactions to sulfites among certain asthmatics. Following the ban on sulfite compounds for washing fresh mushrooms, there have been several efforts to develop wash solutions for use as a suitable replacement for sulfites.

McConnell [70] conducted a review of potential wash additives for mushrooms including sodium hypochlorite, hydrogen peroxide, potassium sorbate, and sodium salts of benzoate, EDTA, and phosphoric acids. The researcher concluded that effective antioxidants, in addition to antimicrobial compounds, were required to enhance shelf life of fresh mushrooms by washing. A fresh mushroom wash solution containing 10,000 ppm hydrogen peroxide and 1000 ppm calcium disodium EDTA was developed. Hydrogen peroxide present in the wash solution acts as a bactericide. Copper is a functional cofactor of the mushroom browning enzyme tyrosinase. EDTA in the wash solution binds copper more readily than tyrosinase, thereby sequestering copper and reducing tyrosinase activity and associated enzymatic browning of mushroom tissue.

Beelman and Duncan [71] developed a mushroom wash process (U.S. Patent 5,919,507). The method employed a first-stage high pH (pH of 9.0 or above) antibacterial wash followed by a neutralizing wash containing browning inhibitors. The neutralizing wash contained a buffered solution of erythorbic acid and sodium erythorbate. Other browning inhibitors such as ascorbates, EDTA, or calcium chloride were identified as suitable ingredients for addition to the neutralizing solution. The process also helped remove debris and delayed microbial spoilage of fresh mushrooms.

Sapers et al. [72] developed a two-stage mushroom wash process employing 10,000 ppm (1%) hydrogen peroxide in the first stage aqueous solution, and 2.25 to 4.5% sodium erythorbate, 0.2% cysteine-HCl, and 500 ppm to 1000 ppm EDTA in aqueous solution in the second stage. The two-stage washing typically yielded mushrooms nearly as white as sulfited mushrooms initially, and whiteness surpassed that of sulfited mushrooms after 1 to 2 days of storage at 12°C [73,74]. The treatment was effective in reducing bacterial populations in wash water and on mushroom surfaces [75] and had minimal effects on mushroom structure and composition [76]. The process was further modified and optimized [72] to include a prewash step using 0.5% (5000 ppm) to 1% (10,000 ppm) hydrogen peroxide. Mushrooms washed by this process were free of adhering soil, less subject to brown blotch than conventionally washed mushrooms, and at least as resistant to enzymatic browning as unwashed mushrooms during storage at 4°C. However, storage at 10° C accelerated development of brown blotch and browning.

6.3.5.3 Irradiation

In 1986 the FDA approved gamma irradiation doses up to 1 kGy on fruits and vegetables for the purpose of insect and/or growth and maturation control. Low-dose gamma irradiation has been reported to be a very effective method of controlling deterioration and improving quality and shelf life of fresh mushrooms [77-79]. Radiation, usually from a cobalt-60 source, is most effective when applied to the mushrooms shortly after harvest. A dose of 1 kGy, an FDA-approved dose, greatly reduced bacterial counts and slowed the rate of senescence [78]. A dose of 0.25 kGy was ineffective in controlling senescence, while 2 kGy showed no significant improvement over 1 kGy in terms of postharvest quality [78]. Cap opening, stipe elongation, surface darkening, and tissue softening were either delayed or prevented by the application of irradiation [78]. Sensory data comparing irradiated mushrooms with unirradiated controls showed that the former had equal or superior flavor and texture scores for both raw and cooked samples [78]. In another study, Ajlouni et al. [14] concluded that low-dose gamma irradiation (1 kGy) was an effective method for improving quality and extending the shelf life of mushrooms under commercial retail conditions, but it would need to be coupled with refrigerated storage to be most effective. Commercial application of irradiation for enhancing the quality of mushrooms has not yet been used in the U.S. However, cultivated mushrooms appear to be a good candidate for irradiation because of their high market value and short shelf life.

Recently, electron-beam irradiation was evaluated for its application to fresh sliced mushrooms [80]. The effects of electron-beam irradiation on microbial counts, color, texture, and enzyme activity of mushroom slices were evaluated at dose levels of 0.5, 1, 3.1, and 5.2 kGy. Irradiation levels above 0.5 kGy reduced total plate counts, yeast and mold, and psychrotrophic bacteria counts to below detectable levels, and prevented microbial-induced browning. Firmness of all samples was similar during storage except for the 5.2 kGy sample. Color was not affected by the irradiation treatments. Electron-beam irradiation at the levels tested did not affect the polyphenol oxidase activity. Irradiation at 1 kGy was most effective in extending shelf life of mushroom slices [80].

6.3.5.4 Pulsed Ultraviolet Light Treatment

Ultraviolet (UV) light is a portion of electromagnetic spectrum ranging from 100 to 400 nm wavelengths. UV light in the wavelength range 100 to 280 nm has germicidal properties due to DNA damage in microorganisms. Several researchers have demonstrated that the UV light can be used for the inactivation of foodborne pathogens without adversely affecting the quality of food. UV light treatment of foods can be accomplished using a pulsed UV system, whereby the energy is stored in a high-power capacitor and is released periodically in short pulses (often in nanoseconds). The pulsed UV light system reduces the temperature buildup as compared to that obtained with a continuous UV light, due to short pulse durations and cooling periods between pulses. Thus, the pulsed UV light process may be considered a nonthermal process.

Beelman et al. [81] conducted an experiment to evaluate the pulsed UV light sterilization system to reduce bacterial populations in/on fresh mushrooms. Pulsed UV light treatment was carried out with a laboratory scale, batch, pulsed light sterilization system (SteriPulse®-XL 3000, Xenon Corporation, Woburn, MA). The system generated 5.6J/cm2 per pulse on the strobe surface for an input voltage of 3800 V and with 3 pulses per second. The output from the pulsed UV light system followed a sinusoidal wave pattern, with 5.6 J/cm2 per pulse being the peak value of the pulse. The pulse width (duration of pulse) was 360 Packed mushrooms were placed in the pulsed UV light sterilization chamber and treated with pulsed light. The first study used a 30-second treatment at a distance of 8 cm from the UV strobe. The control samples did not undergo any pulsed UV treatment. In the second study, treatments with varying treatment time (2 or 4 seconds) and distance from UV strobe (8 or 13 cm) combinations were evaluated. Treated mushrooms were analyzed for total aerobic bacteria, yeast/mold, and coliform populations.

The microbiological results from the first experiment are shown in Table 6.1. The 30-second pulsed UV treatment at 8 cm distance demonstrated a greater than 1 log (90%) reduction for yeast and mold and aerobic bacterial populations. The UV treatment did not significantly affect coliform populations. On visual analysis, the color of the mushrooms as a result of the pulsed UV treatment was negatively impacted due to surface browning.

The microbiological results of the second experiment are depicted in Table 6.2. In general, the UV treatments of 2 or 4 s duration and 8 or 13 cm distance from the UV strobe resulted in 0.9 to 1.6 log reduction in total

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