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Isabelle Babic and Alley E. Watada

A study was undertaken to determine the inhibitory effect of freeze-dried spinach (Spinacia oleracea L.) powder and its native mesophilic aerobic microorganisms (MAM) on the growth of six strains, representing three species, of Listeria in tryptic soy broth (TSB). The cultures were incubated at 10 °C and growth was measured daily. Both spinach powder and MAM inhibited growth; maximum populations at 6 days were 8.8 Log10 colony-forming units (cfu) per mL-1 in control, 6.4 in spinach powder cultures, and 7.4 in MAM cultures. These results indicated that growth of various Listeria species/strains was affected by the bacteriostatic effect of spinach powder and competitive effect of native microorganisms.

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Naoki Yamauchi and Alley E. Watada

Degradation of chlorophyll in spinach (Spinacia olearacea L. cv. Hybrid 612) appeared to be regulated through the peroxidase-hydrogen peroxide pathway, which opens the porphyrin ring, thus resulting in a colorless compound. This conclusion was arrived at from the analysis of chlorophylls (Chls) and their metabolizes by HPLC and of enzyme activities catalyzing the degradative reactions. Chls decreased at 25C but not at 1C. The chlorophyll oxidase pathway was not active, as noted by the lack of accumulation of a reaction product named Chl a-1. Lipid peroxidation increased with storage, but the products of the reaction. did not degrade chlorophyll, as noted by the lack of increase in Chl a-1. Chlorophyllase activity increased, but chlorophyllide, the expected product of the reaction, changed minimally during senescence. Ethylene at 10 ppm did not alter the pathway that degraded chlorophyll in spinach.

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Naoki Yamauchi and Alley E. Watada

Chlorophylls and xanthophylls were monitored in broccoli (Brassica oleracea L. var. italica Plen.) florets stored in air, air + 10 ppm ethylene, or 10% CO2 + 1% O2 controlled atmosphere (CA) at 15 °C. Chlorophylls a and b, as measured with high-performance liquid chromatography, decreased in florets held in air. The decrease was accelerated by ethylene treatment and suppressed in CA. Chlorophyllide a and pheophorbide a were present in fresh broccoli florets, but the levels decreased significantly in all treatments during storage. The oxidized product of chlorophyll a, 132-hydroxychlorophyll a, did not accumulate. Xanthophylls decreased, but new pigments, suggested to be esterified xanthophylls, formed with yellowing in stored florets. The chlorophyll degradative pathway in broccoli florets was not altered by ethylene or CA and differed from that reported for parsley (Petroselium crisum Nym.) and spinach (Spinacia oleracea L.) leaves.

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Hidemi Izumi and Alley E. Watada

Physiology and quality of CaCl2 treated or nontreated `Elite' zucchini squash slices were monitored during storage in air, low O2 (0.25, 0.5 and 1%) or high CO2 (3, 6, and 10%) atmosphere at 10C. O2 consumption and CO2 production were reduced under low O2 and high CO2 atmospheres and the reduction was greater with low O2. C2H4 production was reduced with low O2 and initially with high CO2. After day 2 or 4, C2H4 production under high CO2 increased with the increase being greater at the lower CO2 level. The amount and severity of injury/decay were less under low O2 and high CO2 than air atmosphere. Slices stored under 0.25% O2 atmosphere had less weight loss and injury/decay and greater shear force and ascorbic acid content than those held in air atmosphere. Microbial count, pH, and color were affected by the low O2 only on the last day. CaCl2 had no additive effect.

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Naoki Yamauchi and Alley E. Watada

Pigments in stored parsley leaves (Petroselinum crispum Nym.) were monitored to determine if degradative products of chlorophyll (chl) differed while under different types of atmosphere. The leaves were stored in a closed container under a stream of humidified air at 20C with or without 10 ppm ethylene and with or without 10 percent oxygen and 10 percent carbon dioxide. Analysis of pigments with HPLC showed that chl a and b decreased sharply with or without ethylene and the decrease was considerably less under CA. Chlorophyll a-1, the oxidized form of chl a, was initially low, and the level decreased slightly with all of the storage conditions. Chlorophyllide was also low, but it increased slightly during storage. Xanthophyll derivatives, which appeared to be the esterified xanthophylls, increased slightly during storage. These results indicate that chl degradation in stored parsley leaves was hastened by ethylene or suppressed by CA condition and the pathway of chl degradation did not appear to be altered by the different storage atmospheres.

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Isabelle Babic and Alley E. Watada

The population of Listeria monocytogenes inoculated on lightly processed spinach held at 5C or 10C remained constant for 9 days, whereas the populations of mesophilic aerobic flora, pseudomonas, and enterobacteria increased sharply. We studied the cause of the bacteriostatic activity of spinach leaves against L. monocytogenes. Listeria monocytogenes were inoculated at the concentration of 2 × 104 cells/ml in raw, autoclaved and vacuum filtration sterilized spinach macerates as well as in tryptic soy broth used as control. The concentrations after 24 hat 28C were 2 × 104, 4 × 107, and 4 × 105 cells/ml in respective spinach macerates and 4 × 108 cells/ml in tryptic soy broth. The anti-listeria activity was still present in spinach macerates sterilized by vacuum filtration but not in autoclaved macerates. In conclusion, the bacteriostatic activity against L. monocytogenes might be due to antimicrobial compounds present in spinach leaves or produced by the other microorganisms. Studies are being continued to identify the compounds involved in the anti-listeria activity.

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Isabelle Babic and Alley E. Watada

Studies. were undertaken to investigate the cause of the bacteriostatic activity of spinach leaves against Listeria monocytogenes. Listeria monocytogenes was inoculated at the concentration 104 cells/ml in raw, autoclaved and vacuum filtration sterilized spinach macerates as well as in tryptic soy broth used as control. The concentrations after 24 h at 28C were 105, 5 × l06 and 105 cells/ml in respective spinach macerates and 4 × l08 cells/ml in tryptic soy broth. The anti-Listeria activity was still present in spinach macerates sterilized by vacuum filtration but strongly reduced in autoclaved macerates. Serial dilutions of freeze-dried spinach were tested on the growth of Listeria monocytogenes in tryptic soy broth. The minimum concentration for maximum inhibition was 10 mg·ml–1. The growth of Listeria monocytogenes in tryptic soy broth in mixed cultures with different microbial species isolated from spinach was not inhibited. In conclusion, the bacteriostatic activity against Listeria monocytogenes might be due to antimicrobial compounds present in spinach leaves.

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Isabelle Babic and Alley E. Watada

Fresh-cut spinach has been shown to have a bacteriostatic effect on Listeria monocytogenes ATCC 19111 (Babic et al., 1997). A study was undertaken to determine if this effect is noted on other species of Listeria and to determine if the spinach or the natural microorganisms on the spinach was the cause of the bacteriostatic effect. Six species of Listeria was cultivated in pure tryptic soy broth, tryptic soy broth containing freeze-dried spinach powder, or broth containing mesophilic aerobic microorganisms (MAM) isolated from spinach powder. The cultures were incubated at 10°C for 6 days and growth measured daily. Growth data were analyzed as four factor general linear repeated measures mixed model with species, treatment, and day as the fixed effects. The fixed effects showed a significant interaction between treatment × day and day × species. Results indicated that both the spinach and MAM had an inhibitory effect on Listeria as noted by the maximum population at 6 days, which was 8.8 Log10 cfu/ml in control, 6.4 in spinach powder cultures, and 7.4 in mixed cultures (P < 0.05). Of the six Listeria species, three L. monocytogenes were affected similarly whereas the remaining three, particularly L. innocua, were affected differently. In conclusion, the bacteriostatic effect of fresh-cut spinach differs with Listeria species and the native microorganisms play a major role as competitors.

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Hisashi Kato-Noguchi and Alley E. Watada

Carrot (Daucus carota L.) shreds were stored under a continuous flow of air or 0.5% and 2% O2 (balance N,) for 9 days at 5 and 15C. The resulting changes in respiration, levels of glycolytic intermediates, and activities of ATP: phosphofructo kinase (ATP-PFK), and PPi: phosphofructokinase (PPi-PFK) were monitored. Carrots under low O atmosphere exhibited an increase in RQ due to a greater reduction in 02 consumption than in CO2 production, and the increase in RQ was greater at 0.5% than at 2% O2 at both temperatures. Fructose 1,6-bisphosphate (F1,6P) accumulated with decreased O2 atmosphere and was 2-fold greater at 0.5% than at 2% O2 atmosphere at both temperatures. The levels of other glycolytic intermediates were not significantly influenced by low O2. The increase in PPi-PFK activity occurred at the same time as F1,6P accumulation. A similar relationship was not found with ATP-PFK. These results suggest that PPi-PFK may be involved in regulation of glycolysis under low O2 atmosphere.

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Hidemi Izumi, Alley E. Watada, and Willard Douglas

`Marathon' broccoli (Brassica oleracea L. var. italica) florets were stored in air, low O2(0.25%, 0.5%, and 1 %) or high CO2(3%,6%, and 10%) at 0, 5, and 10C. Oxygen consumption and CO2 production were reduced under low O2 or high CO2atmosphere, the reduction being greater at lower O2 and higher CO2 levels. No differences were found in ethylene production among the different atmospheres. Low O2 and high CO2 retained color of broccoli florets to about the same extent at 10C but had no effect at 0 and 5C. Development of soft rot and browning was suppressed by low O2 or high CO2, but offensive off-odor occurred in 0.25%02 at all temperatures and 0.5% O2 at 10C. These results indicate that the best O2 and CO2 levels seem to be 0.5% O2 and 10% CO2 at 0 and 5C, and 1% O2 and 10% CO2 at 10C.