Apple quality,storage, and washing treatments affect patulin levels in apple cider

Patulin is a mycotoxin produced primarily by Penicillium expansum, a mold responsible for rot in apples and other fruits. The growth of this fungus and the production of patulin are common in fruit that has been damaged. However, patulin can be detected in visibly sound fruit. The purpose of this project was to determine how apple quality, storage, and washing treatments affect patulin levels in apple cider. Patulin was not detected in cider pressed from fresh tree-picked apples (seven cultivars) but was found at levels of 40.2 to 374 microg/liter in cider pressed from four cultivars of fresh ground-harvested (dropped) apples. Patulin was not detected in cider pressed from culled tree-picked apples stored for 4 to 6 weeks at 0 to 2 degrees C but was found at levels of 0.97 to 64.0 microg/liter in cider pressed from unculled fruit stored under the same conditions. Cider from controlled-atmosphere-stored apples that were culled before pressing contained 0 to 15.1 microg of patulin per liter, while cider made from unculled fruit contained 59.9 to 120.5 microg of patulin per liter. The washing of ground-harvested apples before pressing reduced patulin levels in cider by 10 to 100%, depending on the initial patulin levels and the type of wash solution used. These results indicate that patulin is a good indicator of the quality of the apples used to manufacture cider. The avoidance of ground-harvested apples and the careful culling of apples before pressing are good methods for reducing patulin levels in cider.     

Use of hazard analysis critical control point and alternative treatments in the production of apple cider.

The purpose of this study was to evaluate the practices of Maryland cider producers and determine whether implementing hazard analysis critical control point (HACCP) would reduce the microbial contamination of cider. Cider producers (n = 11) were surveyed to determine existing manufacturing practices and sanitation. A training program was then conducted to inform operators of safety issues, including contamination with Escherichia coli O157:H7, and teach HACCP concepts and principles, sanitation procedures, and good manufacturing practice (GMP). Although all operators used a control strategy from one of the model HACCP plans provided, only one developed a written HACCP plan. None developed specific GMP, sanitation standard operating procedures, or sanitation monitoring records. Six operators changed or added production controls, including the exclusion of windfall apples, sanitizing apples chemically and by hot dip, and cider treatment with UV light or pasteurization. Facility inspections indicated improved sanitation and hazard control but identified ongoing problems. Microbiological evaluation of bottled cider before and after training, in-line apples, pomace, cider, and inoculated apples was conducted. E. coli O157:H7, Salmonella, or Staphylococcus aureus were not found in samples of in-line apple, pomace, and cider, or bottled cider. Generic E. coli was not isolated on in-coming apples but was found in 4 of 32 (13%) in-line samples and 3 of 17 (18%) bottled fresh cider samples, suggesting that E. coli was introduced during in-plant processing. To produce pathogen-free cider, operators must strictly conform to GMP and sanitation procedures in addition to HACCP controls. Controls aimed at preventing or eliminating pathogens on source apples are critical but alone may not be sufficient for product safety.     

Microbial levels in Michigan apple cider and their association with manufacturing practices

In recent decades, apple cider has been implicated in a series of outbreaks of foodborne illness. The objective of this study was to determine the presence and concentrations of pathogenic and indicator microorganisms in apple cider processed in Michigan and to evaluate the impact of thermal pasteurization, UV light radiation, and implementation of hazard analysis critical control point (HACCP) plans on these microbes. Cider samples were obtained from Michigan mills between 1997 and 2004 and analyzed for Escherichia coli O157:H7, Salmonella, generic E. coli, total coliforms, and aerobic bacteria. Neither E. coli O157:H7 nor Salmonella were detected in any tested cider samples, suggesting a very low frequency of pathogens in Michigan apple cider. The persistent and relatively high frequency of generic E. coli observed in samples obtained in all years indicates a continued risk of pathogen contamination in Michigan apple cider, especially when it is untreated. The use of thermal pasteurization or UV light radiation and reported implementation of HACCP plans were associated with lower frequency and counts of generic E. coli, total coliforms, and aerobic microorganisms. However, the relatively high counts of indicator organisms in some cider samples that were claimed to be treated according to these pathogen reduction measures indicates that some processors had inadequate practices, facilities, or equipment for pathogen reduction or did not consistently or adequately apply practices or pathogen-reduction equipment in an effective manner.     

Isolation and characterization of Escherichia coli recovered from Maryland apple cider and the cider production environment

Contaminated apple cider has been implicated in several Escherichia coli O157:H7 outbreaks. In an attempt to investigate sources and modes of entry of E. coli into apple cider, samples of fresh apple, pomace, and cider and equipment and mill floor swabs were analyzed for standard plate counts (SPC), total coliforms (TC), fecal coliforms (FC), and E. coli. E. coli was isolated from 14 (33%) of 42 samples of bottled fresh cider, from food equipment in 6 (67%) of 9 mills, and from apples, pomace, or cider in 7 (78%) of 9 mills. Seventy-five E. coli isolates were further characterized for Shiga toxin-producing E. coli (STEC)-associated virulence factors, antimicrobial susceptibility, and pulsed-field gel electrophoresis (PFGE) type. No E. coli O157:H7 or other STEC was identified. Serotyping and PFGE revealed 64 distinct profiles, suggesting that recovered E. coli arose from multiple independent sources. However, on one occasion, E. coli isolated from the source apple sample was closely related to the E. coli identified in the finished cider sample. E. coli isolates were further tested for antimicrobial susceptibility to 17 antimicrobial agents of human and veterinary importance. Fourteen (19%) of the 75 isolates were resistant to at least one of the antimicrobial agents tested, and 9 (12%) were resistant to at least two of these agents. Of the resistant isolates recovered, 64% were resistant to tetracycline and 57% were resistant to streptomycin. Overall, the level of E. coli contamination in source apple samples did not differ significantly from those in samples of pomace, cider at the press, and cider entering the bottling tank; therefore, source apples cannot be dismissed as a potential contributor of E. coli to the cider-making process.     

Influence of fruit variety, harvest technique, quality sorting, and storage on the native microflora of unpasteurized apple cider

Apple variety, harvest, quality sorting, and storage practices were assessed to determine their impact on the microflora of unpasteurized cider. Seven apple varieties were harvested from the tree or the ground. The apples were used fresh or were stored at 0 to 4 degrees C for < or = 5 months and were pressed with or without quality selection. Cider yield, pH, Brix value, and titratable acidity were measured. Apples, postpressing apple pomace, and cider samples were analyzed for aerobic bacteria, yeasts, and molds. Aerobic bacterial plate counts (APCs) of ciders from fresh ground-picked apples (4.89 log CFU/ml) were higher than those of ciders made from fresh, tree-picked apples (3.45 log CFU/ml). Quality sorting further reduced the average APC to 2.88 log CFU/ml. Differences among all three treatment groups were significant (P < 0.0001). Apple and pomace microbial concentrations revealed harvest and postharvest treatment-dependent differences similar to those found in cider. There were significant differences in APC among apple varieties (P = 0.0001). Lower counts were associated with varieties exhibiting higher Brix values and higher titratable acidity. Differences in APC for stored and fresh apples used for cider production were not significant (P > 0.05). Yeast and mold counts revealed relationships similar to those for APCs. The relationship between initial microbial load found on incoming fruit and final cider microbial population was curvilinear, with the weakest correlations for the lowest apple microflora concentrations. The lack of linearity suggests that processing equipment contributed to cider contamination. Tree-picked quality fruit should be used for unpasteurized cider production, and careful manufacturing practices at cider plants can impact both safety and quality of the final product.     

SURVEY OF APPLE GROWING, HARVESTING, AND CIDER MANUFACTURING PRACTICES IN WISCONSIN: IMPLICATIONS FOR SAFETY

To evaluate the safety of current apple growing, harvesting and cider manufacturing practices in Wisconsin, cider manufacturers were contacted in a three-phase survey. Results revealed that seasonal, small-scale production was characteristic of the industry. Most cider mills produced less than 5,000 gal/year; only 6% produced more than 20,000 gal/year. Most cider makers used only tree picked apples (86%), inspected apples before washing (94%), washed (93%) and brushed (87%) apples, but only 16% of mills sanitized washed apples. Most mills (92%) sanitized cider making equipment after each use; however, only a few sanitized between custom pressing apples from different customers. Respondents reported that they strived to improve cider safety by pasteurization (43% of all cider), UV light treatment (4%), use of preservatives (30%), and HACCP (17%). For 31% of all cider, however, processors relied solely on refrigeration and/or freezing. These results show that most cider mills practice many steps believed to enhance cider safety, but results also identify procedures that must be addressed to further improve cider safety.     

Verifying apple cider plant sanitation and hazard analysis critical control point programs: choice of indicator bacteria and testing methods.

The objectives of this study were (i) to evaluate the survival of coliforms, Escherichia coli, and enterococci in refrigerated apple cider; (ii) to develop simple and inexpensive presumptive methods for detection of these bacteria; (iii) to perform a field survey to determine the prevalence of these bacteria on apples and in apple cider; and (iv) based on our results, to recommend the most useful of these three indicator groups for use in verifying apple cider processing plant sanitation and hazard analysis critical control point (HACCP) programs. Eight of 10 coliform strains (5 E. coli, 1 Enterobacter aerogenes, and 2 Klebsiella spp.) inoculated into preservative-free apple cider (pH 3.4, 13.3(o) Brix) survived well at 4 degrees C for 6 days (< or = 3.0 log10 CFU/ml decrease). Of 21 enterococci strains (Enterococcus faecalis, E. faecium, and E. durans), only 2 E. durans and 3 E. faecium strains survived well. Simple broth-based colorimetric methods were developed that detected the presence of approximately 10 cells of coliforms or enterococci. In three field studies, samples of unwashed apples (drops and picked), washed apples, and freshly pressed cider were presumptively analyzed for total coliforms, E. coli, and enterococci using qualitative and/or quantitative methods. Drop apples were more likely than picked apples to be contaminated with E. coli (26.7% vs. 0%) and enterococci (20% vs. 0%). Washing had little effect on coliform populations and in one field study was associated with increased numbers. Total coliform populations in cider ranged from < 1 CFU/ml to > 738 most probable number/ml, depending on the enumeration method used and the sample origin. E. coli was not recovered from washed apples or cider, but enterococci were present on 13% of washed apple samples. The qualitative coliform method successfully detected these bacteria on apples and in cider. Based on its exclusively fecal origin, good survival in apple cider, and association with drop apples, we conclude that E. coli is the most useful organism for verifying apple cider sanitation and HACCP programs.     

Efficacy of ultraviolet light for reducing Escherichia coli O157:H7 in unpasteurized apple cider

This study examined the efficacy of UV light for reducing Escherichia coli O157:H7 in unpasteurized cider. Cider containing a mixture of acid-resistant E. coli O157:H7 (6.3 log CFU/ml) was treated using a thin-film UV disinfection unit at 254 nm. Dosages ranged from 9,402 to 61,005 microW-s/cm2. Treatment significantly reduced E. coli O157:H7 (P < or = 0.0001). Mean reduction for all treated samples was 3.81 log CFU/ml. Reduction was also affected by the level of background microflora in cider. Results indicate that UV light is effective for reducing this pathogen in cider. However, with the dosages used in this experiment, additional reduction measures are necessary to achieve the required 5-log reduction.     

DIPHENYLAMINE RESIDUES IN APPLES AND APPLE CIDER

Diphenylamine (DPA) is an antioxidant which is widely used on apples for scald control during fruit storage. Since appreciable residues of DPA remain on apples when sold, it was of interest to study the extent of possible transfer of the compound to cider. Cider was expressed from five cultivars ofDPA-treated apples following their storage and the original fruit, the pomace and cider was analyzed for DPA residues. Only traces of the compound were found in cider with virtually all of the DPA found concentrated in the pomace. The toxicologic significance of the results are discussed.     

Potential sources of microbial contamination in unpasteurized apple cider

A study was conducted to identify possible sources of microbial contamination and to assess the effect of good cleaning and sanitation practices on the microbial quality and safety of unpasteurized apple cider. Raw unwashed apples, washed apples, cleaning water, fresh cider, and finished cider samples were collected from five Ontario producers over 4 months and microbiologically tested. Total coliforms were found in 31, 71 and 38% of the unwashed apple, water, and washed apple samples, respectively. Escherichia coli was found in 40% of the water samples from one producer alone. The washing step was identified as a potential source of contamination, possibly due to water in the dump tanks seldom being refreshed, and because scrubbers, spray nozzles, and conveyors were not properly cleaned and sanitized. Higher total coliform counts (P < 0.0001) and prevalence (P < 0.0001) in fresh cider compared with those in unwashed apples and washed apples indicated considerable microbial buildup along the process, possibly explained by the lack of appropriate equipment sanitation procedures. Results showed that producers who had better sanitary practices in place had lower (P < 0.001) total coliform prevalence than the rest of the producers. Overall results show that good sanitation procedures are associated with improved microbial quality of fresh cider in terms of total coliforms and that operators who pasteurize and/or UV treat their product should still be required to have a sound good manufacturing practices program in place to prevent recontamination. Cryptosporidium parvum, an important pathogen for this industry, was found in different sample types, including washed apples, water, and fresh and finished cider.     

Flavor Characteristics of Irradiated Apple Cider During Storage: Effect of Packaging Materials and Sorbate Addition

ABSTRACT: Apple cider, with (0.1%) and without potassium sorbate, was packaged in 3 different materials to evaluate the effects of irradiation (2 kGy) and storage on flavor. Irradiated apple cider samples were compared with unirradiated samples stored in glass jars. Volatile flavor compounds, soluble solids, and titratable acidity were determined weekly throughout 3 wk of refrigerated storage. Oxygen permeability of the packaging materials was important in the retention of flavor during storage. Cider irradiated and stored in polystyrene containers or nylon-6 packaging materials (low oxygen permeability) had lower rates of loss of characteristic flavor compounds compared with unirradiated apple cider and cider irradiated and stored in low-density polyethylene (high oxygen permeability). The presence of sorbate, functioning as a yeast and mold inhibitor, reduced the rates of loss of the characteristic flavor compounds and the fermentation of sugars to acids. Principal component analysis resulted in several esters characteristic of apple flavor, soluble solids, and titratable acidity loading onto the 1st principal component (PC-1). Packaging material and sorbate treatment had the greatest effect on the compounds that loaded onto PC-1.     

Malolactic Fermentation as a Technique for the Deacidification of Hard Apple Cider

ABSTRACT:  Malolactic fermentation (MLF), the conversion of malate to lactate, is an important process leading to the deacidification of hard apple cider. MLF is dependent on the levels of inhibitory factors such as sulfur dioxide and ethanol. To assess the effect of these 2 factors on MLF, hard apple cider was produced from pasteurized, unfiltered apple cider ( Malus domestica  cvs Red Delicious, Golden Delicious, Braeburn, and Fuji). Apple cider was treated with 2 levels of sulfur dioxide (50 and 80 ppm) and then fermented using  Saccharomyces cerevisiae  montrachet. After the primary fermentation, 1 set of the samples remained unadjusted and 100% ethyl alcohol was used to adjust other sets of samples to 7%, 9%, or 11% (v/v) ethanol. Following the ethanol adjustment,  Oenococcus oeni  MCW was used to initiate the MLF in half of the samples. Cider parameters monitored throughout the fermentations included organic acid content, titratable acidity, pH, ethanol production, and sugar content. Since samples containing either sulfur dioxide level had similar sugar utilization rates and ethanol production it was concluded that sulfur dioxide had no effect on the primary fermentation. Sulfur dioxide content was shown to have an impact on MLF. There was no difference in the rate of malic acid consumption, but lactic acid production was faster in the 50-ppm sulfur dioxide samples. MLF was not inhibited by ethanol content.     

A study of U.S. orchards to identify potential sources of Escherichia coli O157:H7

The association of unpasteurized apple cider with Escherichia coli O157:H7 foodborne illness has led to increased interest in potential reservoirs of this pathogen in the orchard. Fourteen U.S. orchards were surveyed in autumn 1999 to determine the incidence and prevalence of E. coli O157:H7, E. coli, total aerobic microflora, and yeasts and molds. Fruit samples (n = 63) (eight apple and two pear varieties) and soil, water, and fecal samples were collected. Samples were plated on (i) tryptic soy agar for total mesophilic aerobic count, (ii) E. coli and coliform Petrifilm for total coliforms and E. coli, and (iii) yeast and mold Petrifilm. Samples positive for coliforms and E. coli were enriched and tested for E. coli O157:H7. Fruit was also tested for internalization of microflora by aseptically removing the core, stem, and calyx areas, and the individual sections were assessed for the categories of microflora listed above. E. coli was detected in soil and water and in 6% of fruit samples (three pear samples and one apple sample), generally collected from areas previously designated as high risk in this study. However, no E. coli O157:H7 was found. Coliforms were found in 74% of fruit samples and were internalized in the cores of 40% of fruit tested. Yeasts and molds were internalized in 96.7% of samples and aerobic bacteria in 89.6%. E. coli was not found to be internalized. Total aerobic counts and total coliforms were higher in dropped and damaged fruit (P < 0.05). Findings suggest that dropped or damaged fruit should not be included in fruit designated for the production of unpasteurized juice or for the fresh or fresh-cut market. In addition, orchards should be located away from potential sources of contamination, such as pastures.     

Location of Escherichia coli O157:H7 on and in apples as affected by bruising, washing, and rubbing

Confocal scanning laser microscopy (CSLM) was used to determine the location of Escherichia coli O157:H7 cells on the surface and in tissue of bruised Red Delicious cv. apples. Undamaged and bruised apples were inoculated by immersing in a suspension of E. coli O157:H7 cells transformed with a plasmid that encodes for the production of a green fluorescent protein. Apples were then washed in 0.1% (wt/vol) peptone water and/or rubbed with a polyester cloth and examined to determine if these treatments removed or introduced cells into lenticels, cutin, and cracks on the skin surface. Optical slices of the apples obtained using CSLM were examined to determine the depth at which colonization or attachment of cells occurred. Populations of E. coli O157:H7 on the surface of apples were determined to assess the effectiveness of washing and rubbing in physically removing cells. The location of cells on or in undamaged and bruised areas of apples that were not washed or rubbed did not differ significantly. However, washing apples resulted in an approximate 2-log reduction in CFU of E. coli O157:H7 per cm2 of apple surface. On unwashed apples, cells were detected at depths up to 30 microm below the surface. No E. coli O157:H7 cells were detected at locations more than 6 microm below the surface of washed apples. Cells that remained on the surface of rubbed apples appeared to be sealed within naturally occurring cracks and crevices in waxy cutin platelets. These cells may be protected from disinfection and subsequently released when apples are eaten or pressed for cider production.     

Inactivation of Escherichia coli O157:H7 and other naturally occurring microorganisms in apple cider by electron beam irradiation

Two Escherichia coli O157:H7 strains, SEA 13 B88 gfp 73ec and B6-914 gfp 90ec, together with two bacteria, three yeasts, and two molds that were randomly selected from a collection of microorganisms found on apples or in apple cider, were inoculated into apple cider and subjected to electron beam irradiation at several doses between 0.0 and 2.3 kGy at the Iowa State University Linear Accelerator Facility. The D-values for the E. coli O157:H7 strains ranged between 0.25 and 0.34 kGy; the D-values for most of the normal flora from apples ranged between 0.24 and 0.59 kGy. By taking into account possible variations in treatment conditions, it was calculated that irradiation at 2.47 kGy should achieve a 5-log reduction of E. coli O157:H7 in apple cider at the 95% confidence level. Naturally occurring yeasts might survive such irradiation treatment.     

Efficacy of washing with a commercial flatbed brush washer, using conventional and experimental washing agents, in reducing populations of Escherichia coli on artificially inoculated apples

Conventional and experimental washing formulations were applied with a commercial flatbed brush washer under conditions representative of commercial practice to determine their efficacy in decontaminating apples inoculated with a nonpathogenic Escherichia coli strain. Golden Delicious apples (18 kg) inoculated with E. coli were mixed with approximately 109 kg of uninoculated Fuji apples (distinctly different in appearance) in a wet dump tank containing 1,325 liters of water at 20 degrees C for 15 min. The combined apples were washed in a flatbed brush washer with the following washing solutions: water at 20 degrees C, water at 50 degrees C, 200 ppm of chlorine (pH 6.4) at 20 degrees C, 8% trisodium phosphate at 20 degrees C, 8% trisodium phosphate at 50 degrees C, 5% hydrogen peroxide at 20 degrees C, 5% hydrogen peroxide at 50 degrees C, 1% APL Kleen 245 at 50 degrees C, and two-stage washing treatments using the combination of 1% APL Kleen 245 at 20 or 50 degrees C followed by 5% hydrogen peroxide at 35 or 50 degrees C. None of the washing treatments tested under the conditions of this experiment significantly reduced the E. coli populations on the inoculated apples or in cider made from these apples, probably as a consequence of the inability of this washing system to inactivate or remove the bacterial cells in inaccessible calyx and stem areas of apples. These results are important because they demonstrate the need for new fruit washing technology that can overcome this limitation. Also, there was no significant cross-contamination of the Fuji apples in the dump tank. Significant cross-contamination of cider, made with uninoculated apples, occurred in the hammer mill and/or the press cloth when these units were not sanitized following a trial with inoculated apples.     

Characterization of Apples and Apple Cider Produced by a Guelph Area Orchard

Thermal stability of food-borne pathogens in apple cider is influenced by the composition of the product. As a preliminary step to determine the effect of pasteurization of apple cider on the survival of Escherichia coli O157:H7, a study was carried out to characterize apples and unpasteurized apple cider produced by a Guelph area orchard. Samples of commercial unpasteurized cider and the constituent apples were collected over 13 wk from August to November 1998, and unpasteurized laboratory cider was made from the individual apple varieties. pH, titratable acidity, turbidity, total microbial counts, total solids and °Brix for filtered and unfiltered samples were measured. The maximum, minimum, and average values for all unpasteurized commercial cider samples were found as follows: pH, 3.71, 3.17, and 3.43; titratable acidity, 93.47, 49.46, and 69.95 mL of 0.1 N NaOH/100 mL; total solids, 13.21, 10.93, and 11.90%; °Brix, 13.01, 11.17, and 12.02; turbidity, 238.1, 145.1, and 204.9 nephelometric turbidity units; and total plate count, 4.91, 2.61, 3.75 log cfu/mL. There were no significant differences (P>0.05) between filtered and unfiltered samples. In addition, in commercial unpasteurized cider, there were no significant differences (P>0.05) with respect to any of the factors with the time of processing. The composition of the unpasteurized laboratory cider made from individual apple varieties was dependent on the variety, but was generally within the ranges from the published literature values. McIntosh apples showed a significant (P\le0.05) decrease in titratable acidity with time of harvest. The results suggest that it is necessary to take the composition of commercial apple cider into account when developing thermal inactivation models for food-borne pathogens.     

UV inactivation of bacteria in apple cider

Apple cider, inoculated with Escherichia coli and Listeria innocua, was processed using a simple UV apparatus. The apparatus consisted of a low-pressure mercury lamp surrounded by a coil of UV transparent tubing. Cider was pumped through the tubing at flow rates of 27 to 83 ml/min. The population of E. coli K-12 was reduced by 3.4 +/- 0.3 log after being exposed for 19 s at a treatment temperature of 25 degrees C. The population of L. innocua, which was more resistant to UV, was reduced by 2.5 +/- 0.1 log after being exposed for 58 s. The electrical energy for the process was 34 J/ml and is similar to that for conventional thermal processing. UV processing has the potential to improve the safety and extend the shelf life of apple cider.     

CIDER PRODUCTION WITH IMMOBILIZED LEUCONOSTOC OENOS

Cider obtained with Saccharomyces cerevisiae and immobilized Leuconostoc oenos attained the desired attenuation without residual sugars. Decarboxylation of L-malic to L-lactic acid by heterolactic bacteria and the formation of secondary products in cider were studied using immobilized cells of L. oenos IC11. Malic acid metabolism was influenced by the temperature at which the fermentation took place. The production of ethyl acetate and methanol was influenced by the type of L. oenos inoculation. Acetaldehyde was the most discriminating variable in the ciders obtained at different temperatures. The use of multivariate analysis allowed differentiation of the ciders obtained on the basis of the inoculation model followed .