Viability of sublethally injured indicator and pathogenic coliform bacteria on fresh-cut cabbage during storage in an active MAP of 10% CO2.

Shredded cabbage treated with either tap water or electrolyzed water was stored in an active modified atmosphere packaging (MAP) of 10% CO2 for 5 d at 10℃, 7 d at 5℃, and 8 d at 1℃ to evaluate the occurrence and viability of sublethally injured coliform bacteria. The CO2 and O2 concentrations in the packages approached an equilibrium of 10% CO2 and 10% O2 during storage at all temperatures tested. Coliforms in shredded cabbage increased during storage at all three temperatures, with the increase being greater at 10℃. Sublethal injury at 65% to 69% for the coliforms was detected only on cabbage samples treated with electrolyzed water and stored at 5℃ for 4 and 7 d. Enterobacter cloacae was one of the injured species of coliform bacteria in shredded cabbage. Shredded cabbage was inoculated with chlorine-injured Escherichia coli O157:H7 and stored at 5℃ for 6 d in an active MAP of 10% CO2. Counts of E. coli O157:H7 remained almost constant during storage, and injured E. coli O157:H7 ranging from 50% to 70% were found on shredded cabbage throughout the storage period. These results indicate that sublethally injured indicator and pathogenic bacteria would be found on fresh-cut cabbage in the realistic MAP storage at 5℃.

Viability of sublethally injured bacteria of fresh and fresh-cut vegetables from the field through distribution.

Bacterial stresses can occur from the production to the distribution environments of produce, and these stresses can lead to nonlethal bacterial damage that is an injured state called sublethally injured bacteria. The damage is mainly due to the disruption of the surface structure and cytoplasmic membrane of the cells. Sublethally sanitizer-injured indicator coliform bacteria injured by chlorine, ethanol, and/or fungicide stress could exhibit on vegetables during production and harvest. Chlorine stress and cold stress could induce sublethally injured indicator and pathogenic coliform bacteria on fresh-cut vegetables during processing and subsequent storage. Enterobacter kobei and Pantoea ananatis injurd by chlorine stress, E. amnigenus, E. asburiae, and E. kobei injured by ethanol stress, and Rahnella aquatilis, Yersinia mollaretii, and Escherichia coli injured by fungicide stress could be amongst the injured cells in the coliforms detected in the produce environments. To ensure the microbiological quality and safety of fresh-cut vegetables, it is necessary to adjust the concentration of sanitizer to a level that kills bacteria and does not produce sanitizer- injured cells when sanitizer is applied to the produce, and also to consider the storage temperature to inhibit the recovery of injured bacteria due to cold injury during the chilling storage period.

Influence of Artificial Inoculation with Pseudomonas fluorescens on Enzymatic Browning Reactions of Fresh-cut Potatoes.

We initially correlated fluorescent pseudomonads and severity of enzymatic browning on fresh-cut potatoes. Subsequently, we determined the influence of inoculation with Pseudomonas fluorescens following its isolation from the brown tissues on the browning response on fresh-cut potatoes. Bacterial counts on potato slices were higher on browning tissues than on non-browning tissues. P. fluorescens that has been isolated only from the severely browning tissues developed brown discoloration on surface tissues when inoculated onto potato slices. When potato slices were initially inoculated with 103 colony-forming unit (CFU) per mL of P. fluorescens and then stored at 5ºC, bacterial counts, polyphenol oxidase (PPO) activity, phenolic content, and browning severity increased after 3 days of storage. We observed plant PPO derived from potatoes and bacterial PPO released by P. fluorescens and dictated that the plant PPO contributed to browning reactions because only the plant PPO was activated at pH 6-7 that lies in potato tissues. The PPO1 gene that contributed to browning on potatoes was expressed prominently in potato tissues following inoculation with P. fluorescens. These results indicated that P. fluorescens enhanced browning of fresh-cut potatoes by inducing the plant PPO gene, plant PPO activity, and accumulation of phenolics as a biocontrol agent.

Viability of Chlorine-injured Escherichia Coli O157:H7 on Fresh-cut Cabbage during Cold Storage in High CO2 Atmospheres.

Viability of chlorine-injured E. coli O157:H7 inoculated onto shredded cabbage was evaluated during storage in air or high CO2 controlled atmospheres (CA) of 5%, 10%, and 15% at 10℃ and in a modified atmosphere packaging (MAP) at 5℃ and 10℃. When shredded cabbage was inoculated with chlorine-injured E. coli O157:H7 (% injury = 65%) and then stored in air or CA at 10℃, counts of E. coli O157:H7 increased during storage and injured E. coli O157:H7 (% injury = 34-66%) were detected on samples throughout the storage regardless of the CO2 atmosphere. When shredded cabbage inoculated with chlorine injured E. coli O157:H7 (% injury = 45-59%) were stored in a MAP using either a high or low oxygen transmission permeability (OTR) package film, the counts of E. coli O157:H7 increased during storage at 10℃ and they remained constant during storage at 5℃. Injured E. coli O157:H7 were detected on shredded cabbage at a 54-56% level in a low OTR film at 10℃ and a 73-74% level in a high OTR film at 5℃. These results indicated that chlorine-injured E. coli O157:H7 inoculated on fresh-cut cabbage exhibited different degrees of injury during storage in a high CO2 CA and MAP at 5℃ or 10℃.

Viability of sublethally injured coliform bacteria on fresh-cut cabbage stored in high CO2 atmospheres following rinsing with electrolyzed water.

The extent of sublethally injured coliform bacteria on shredded cabbage, either rinsed or not rinsed with electrolyzed water, was evaluated during storage in air and high CO2 controlled atmospheres (5%, 10%, and 15%) at 5°C and 10°C using the thin agar layer (TAL) method. Sublethally injured coliform bacteria on nonrinsed shredded cabbage were either absent or they were injured at a 64-65% level when present. Rinsing of shredded cabbage with electrolyzed water containing 25ppm available chlorine reduced the coliform counts by 0.4 to 1.1 log and caused sublethal injury ranging from 42 to 77%. Pantoea ananatis was one of the species injured by chlorine stress. When shredded cabbage, nonrinsed or rinsed with electrolyzed water, was stored in air and high CO2 atmospheres at 5°C for 7days and 10°C for 5days, coliform counts on TAL plates increased from 3.3-4.5 to 6.5-9.0 log CFU/g during storage, with the increase being greater at 10°C than at 5°C. High CO2 of 10% and 15% reduced the bacterial growth on shredded cabbage during storage at 5°C. Although injured coliform bacteria were not found on nonrinsed shredded cabbage on the initial day, injured coliforms at a range of 49-84% were detected on samples stored in air and high CO2 atmospheres at 5°C and 10°C. Injured cells were detected more frequently during storage at both temperatures irrespective of the CO2 atmosphere when shredded cabbage was rinsed with electrolyzed water. These results indicated that injured coliform bacteria on shredded cabbage, either rinsed or not rinsed with electrolyzed water, exhibited different degrees of injury during storage regardless of the CO2 atmosphere and temperature tested.

Enumeration and Identification of Coliform Bacteria Injured by Chlorine or Fungicide Mixed with Agricultural Water.

Chemical sanitizers may induce no injury (bacteria survive), sublethal injury (bacteria are injured), or lethal injury (bacteria die). The proportion of coliform bacteria that were injured sublethally by chlorine and fungicide mixed with agricultural water (pond water), which was used to dilute the pesticide solution, was evaluated using the thin agar layer (TAL) method. In pure cultures of Enterobacter cloacae , Escherichia coli , and E. coli O157:H7 (representing a human pathogen), the percentage of chlorine-injured cells was 69 to 77% for dilute electrolyzed water containing an available chlorine level of 2 ppm. When agricultural water was mixed with electrolyzed water, the percentage of injured coliforms in agricultural water was 75%. The isolation and identification of bacteria on TAL and selective media suggested that the chlorine stress caused injury to Enterobacter kobei . Of the four fungicide products tested, diluted to their recommended concentrations, Topsin-M, Sumilex, and Oxirane caused injury to coliform bacteria in pure cultures and in agricultural water following their mixture with each pesticide, whereas Streptomycin did not induce any injury to the bacteria. The percentage of injury was 45 to 97% for Topsin-M, 80 to 87% for Sumilex, and 50 to 97% for Oxirane. A comparison of the coliforms isolated from the pesticide solutions and then grown on either TAL or selective media indicated the possibility of fungicide-injured Rahnella aquatilis , Yersinia mollaretii , and E. coli . These results suggest the importance of selecting a suitable sanitizer and the necessity of adjusting the sanitizer concentration to a level that will kill the coliforms rather than cause sanitizer-induced cell injury that can result in the recovery of the coliforms.

Use of Pressure for Improving Storage Quality of Fresh-Cut Produce.

The microflora of fresh-cut produce is comprised primarily of phytopathogenic and soilborne organisms, but the product could be contaminated with foodborne pathogens. Populations of bacteria, molds, and yeasts associated with fresh-cut produce decreased to non-detectable levels following a high pressure (HP) treatment of 400 MPa for 10 min at room temperature, except for spore-forming bacteria such as Bacillus spp. which were inactivated when subjected to 600 MPa at 60 °C for 10 min. The HP treatment of 400 MPa for 5-10 min at room temperature for fresh-cut lotus root and pineapple may be commercially feasible as an alternative to chemical sterilization and thermal blanching, respectively. The HP treatment reduced the epiphytic microorganisms of the products to non-detectable levels, and the microbial counts remained at the initial levels during storage at 1 °C with minimal changes in physicochemical and visual quality of the products. However, the HP treatment induced cellular disruption in plant tissue that contributed to the changes in appearance of several fresh-cut vegetables. To improve storage quality, combining lower pressures with complementary technologies should be useful for successful application of HP for other fresh-cut produce.

Changes in bacterial flora of Japanese cabbage during growth and potential source of flora.

Bacterial flora of cabbage were identified and enumerated during various stages of growth, and the potential sources of contamination in the field were determined. Bacterial counts increased from below the level of detection (2.4 log CFU/g) on seeds to 2.5 to 5.7 log CFU/g on seedlings. After transplanting, the counts of mesophilic aerobic bacteria on leaves decreased and then increased to 5.7 log CFU/g on outer leaves, 5.0 log CFU/g on middle leaves, and 3.0 log CFU/g on inner leaves at the harvesting stage. Counts of coliforms were below the level of detection during the growing period of the leaves. Bacteria isolated from cabbage seeds, seedlings, and leaves were soilborne organisms such as Bacillus, Curtobacterium, and Delftia and phytopathogenic organisms such as Pseudomonas, Pantoea, and Stenotrophomonas. These bacteria were found frequently in seeding machines, potting soil mix, soil, agricultural water, pesticide solutions mixed with the agricultural water, liquid fertilizers, and chemical fertilizers. Contamination from these environmental sites occurred throughout the cabbage growing period rather than only at the harvesting stage. These results indicate that use of clean water for irrigation and for mixing with pesticides and amendments from seeding to the harvesting stage is an important part of a good agricultural practices program for cabbage in Japan.

Potential sources of microbial contamination of satsuma mandarin fruit in Japan, from production through packing shed.

Potential sources of microbial contamination of satsuma mandarin fruit were investigated from production through the packing shed in the 2005 season. Microbial counts in the peel and flesh during the fruit development stage were below 2.4 log CFU/g for bacteria and 3 log CFU/g for fungi, except for the peel in August and September. In the field environment, the highest microbial counts were found in fallen leaves on the ground, followed by soil, organic fertilizer, and agricultural water. Only the pesticide solution collected in July was positive for Salmonella, while no verotoxin-producing Escherichia coli was detected from any of the samples. The bacterial and mold flora in the peel comprised phytopathogenic organisms such as bacteria genus Pantoea and mold genus Mycosphaerella and soilborne organisms such as bacteria genus Bacillus and mold genus Cladosporium, which were found in soil, fallen leaves, agricultural water, and cloth mulch throughout the production season. After fruit harvest and sorting, microbial counts of the peel increased, while those of the flesh remained below the lower limit of detection. Although some of the preharvest sources could also be postharvest sources, some packing shed equipment was assumed to be postharvest sources, because Bacillus cereus was not identified from the fruit in the production field but was detected on the peel after sorting and on equipment such as gloves, plastic harvest basket, and size sorter. These results suggest that using sanitizers for agricultural water and packing sheds to prevent cross-contamination would be useful in a good agricultural practices program of the satsuma mandarin in Japan.

On-farm sources of microbial contamination of persimmon fruit in Japan.

Potential sources of microbial contamination for persimmon fruit during growing and harvesting in the 2005 season were investigated to provide a baseline to design the good agricultural practices program for persimmons in Japan. Microbial counts in the peel of persimmon fruit during production season were close to or below 2.4 log CFU/g for bacteria and 3.0 log CFU/g for fungi but were above these values on harvested fruit. The counts in the flesh were below the detection level with all fruit. Bacteria and molds isolated from peel and flesh of persimmons during growing were phytopathogenic and soilborne organisms such as bacteria genera Enterobacter and Bacillus and mold genera Fusarium and Cladosporium, which were found in soil, weeds, agricultural water, and pesticide solution throughout the production season. The agricultural water was one of the most important potential preharvest sources, because Escherichia coli O157:H7 was identified from agricultural water in May, and Salmonella was detected in agricultural water, pesticide solution containing the agricultural water for the mixture, and soil after application of the pesticide solution in June. Neither of the two pathogenic bacteria was detected in any of the fruit samples. Microbial counts and diversity in the peel of persimmons at harvest increased after contact with plastic harvest basket and container, which could be sources of contamination during harvesting. Therefore, monitoring and management on-farm should focus on agricultural water and harvest equipment as important control points to reduce microbial contamination on persimmons.