Survival and growth of Enterobacter sakazakii on fresh-cut fruits and vegetables and in unpasteurized juices as affected by storage temperature

Enterobacter sakazakii is an emerging foodborne pathogen that has caused illnesses and deaths in infants and elderly immunocompromised adults. Outbreaks of E. sakazakii infection have been associated with infant formulas, but the documented presence of this pathogen in a wide variety of ready-to-eat foods, including lettuce and other raw vegetables, makes it important to learn more about its behavior in these environments. We investigated the survival and growth characteristics of E. sakazakii on fresh-cut apple, cantaloupe, strawberry, watermelon, cabbage, carrot, cucumber, lettuce, and tomato and in juices prepared from these fruits and vegetables. Produce and juices were inoculated with E. sakazakii at 2 to 3 log CFU/g and 1 to 2 log CFU/ml, respectively, and stored at 4, 12, or 25 degrees C. Populations either did not change or gradually decreased in fresh-cut produce and juices stored at 4 degrees C but grew at 12 degrees C on fresh-cut apple, cantaloupe, watermelon, cucumber, and tomato and in all juices except apple, strawberry, cabbage, and tomato juices. All fresh-cut fruits and vegetables except strawberry supported growth of E. sakazakii at 25 degrees C. Growth occurred in all juices except apple, strawberry, and cabbage juices, followed by decreases in population to < 1 CFU/ml after 48 to 72 h, which coincided with decreases in pH and an increase in the population of lactic acid bacteria. Increases in total counts occurred in all juices except strawberry juice stored at 25 degrees C and apple and strawberry juices stored at 12 degrees C. Total counts increased in cantaloupe, carrot, cucumber, and lettuce juices stored at 4 degrees C. Populations of molds and yeasts increased in apple and tomato juices stored at 25 degrees C but decreased to <1 CFU/ml in cabbage, lettuce, and cucumber juices. Further characterization of the behavior of E. sakazakii on fresh produce and in unpasteurized juice as affected by commercial packaging and handling practices is needed.     

Chemical characterization of fruit and fungal volatiles attractive to dried-fruit beetle,Carpophilus hemipterus (L.) (Coleoptera: Nitidulidae)

The chemical basis underlying orientation to fruit and fungal odors was investigated for the dried-fruit beetle,Carpophilus hemipterus (L.). In wind-tunnel bioassays of walking and flight response from 1.8 m, beetles were attracted to odors of the yeastSaccharomyces cerevisiae on agar, aseptic banana, or banana inoculated withS. cerevisiae, although both banana substrates elicited greater response than the yeast alone. When presented in a two-choice bioassay, the yeast-inoculated banana attracted approximately twice as many beetles as did the aseptic banana. GC-MS analysis of the headspace volatiles above these odor sources revealed a somewhat more complex and concentrated volatile profile for yeast-inoculated banana than for aseptic banana. The odor from yeast on agar had fewer components, and these were present at lower concentrations than the odors of either banana substrate. By blending mineral-oil or aqueous solutions of the 18 components of inoculated-banana odor in varying concentrations, it was possible to mimic closely the headspace profile of the natural odor. This synthetic odor also elicited beetle attraction in the wind tunnel at levels comparable to the inoculated banana. Through a series of bioassays in which individual components were subtracted from or added to a synthetic odor blend, it was determined that ethyl acetate, acetaldehyde, 2-pentanol, and 3-methylbutanol comprised the simplest blend of compounds evoking full behavioral response. However, 2-methylpropanol or butanol were apparently interchangeable with 3-methylbutanol in this blend, and comparable response could also be elicited by replacing acetaldehyde with a combination of both 2-pentanone and 3-hydroxy-2-butanone. Thus, our results suggest that this generalist insect herbivore locates its hosts by a long-range response to a variety of blends of common fruit volatiles, whose concentrations are enhanced by fungi.     

Characterization of chemicals mediating ovipositional host-plant finding byAmyelois transitella females

Ovipositional host-finding in the navel orangeworm,Amyelois transitella (Walker), is brought about by an in-flight response to host odors. Wind-tunnel studies of the response of gravid females to almonds showed that this response is mediated primarily by long-chain fatty acids, particularly oleic acid and linoleic acid. Evidence for the behavioral activity of fatty acids is based on the fact that: (1) behavioral activity of almond oil was concentrated in a single liquid chromatographic fraction whose composition was predominantly long-chain fatty acids, (2) behavioral activity was lost when either almond oil or the active fraction of that oil was treated with diazomethane, (3) full activity was elicited by a selective extraction of free fatty acids from crude almond oil, and (4) upwind response by females was elicited by a blend of synthetic oleic and linoleic acids, albeit at a level less than that elicited by almond oil. Five fatty acids identified from the almond oil were: myristic acid (1%), palmitic acid (16%), stearic acid (3%), oleic acid (58%), and linoleic (22%). Attraction to various combinations of synthetic acids was observed only when oleic acid was present, and oleic acid elicited upwind flights to the source when presented alone; however, short-range responses were enhanced by the addition of linoleic acid, which elicited no long-range orientation by itself. Despite significant levels of attraction to synthetic blends, the percentage of females flying to the source was lower than that flying to acidulated almond oil, the best natural attractant tested. Thus, although longrange response may be mediated primarily by a blend of oleic and linoleic acids, additional and as yet unidentified components must also play an important role. Long-range chemically modulated host finding in this and other generalist plant feeders is discussed with respect to current models of the evolution of host finding, and it is argued that suggestions that long-range host finding should be correlated with narrowness of host utilization are logically flawed and are not supported by our current understanding of specific examples of host finding.     

Taxon-specific differences in responsiveness to capsaicin and several analogues: Correlates between chemical structure and behavioral aversiveness

The present set of experiments was designed to explore avian insensitivity to capsaicin. Based upon a molecular model of avian chemosensory repellency, we hypothesized that structural modifications of the basic capsaicin molecule, which is itself not aversive to birds, might produce aversive analogues. To this end, European starlings (Sturnus vulgaris) and Norway rats (Rattus norvegicus) were given varied concentrations of synthetic capsaicin and four analogues (methyl capsaicin, veratryl amine, veratryl acetamide, vanillyl acetamide) in feeding and drinking tests. The results agreed with a model that we are developing to describe the chemical nature of avian repellents. Synthetic capsaicin and vanillyl acetamide were not repellent to birds, owing to the presence of an acidic phenolic OH group. Conversely, veratryl acetamide was aversive, due to the basic nature of this compound. For rats, repellent effectiveness among compounds was reversed: synthetic capsaicin was the best repellent while veratryl acetamide was the worst. We speculate that this taxonomic reversal may reflect basic differences in trigeminal chemoreception. In any case, it is clear that chemical correlates of mammalian repellents are opposite to those that predict avian repellency.     

Role of lipid structure in the activation of phospholipase A2 by peroxidized phospholipids

The time course of hydrolysis of a mixed phospholipid substrate containing bovine liver 1,2-diacyl-sn-glycero-3-phosphocholine (PC) and 1,2-diacyl-sn-glycero-3-phosphoethanolamine (PE) catalyzed byCrotalus adamanteus phospholipase A₂ was measured before and after peroxidation of the lipid substrate. The rate of hydrolysis was increased after peroxidation by an iron/adenosine diphosphate (ADP) system; the presence of iron/ADP in the assay had a minimal inhibitory effect. The rate of lipid hydrolysis was also increased after the substrate was peroxidized by heat and O₂. Similarly, peroxidation increased the rate of hydrolysis of soy PC liposomes that did not contain PE. In order to minimize interfacial factors that may result in an increase in rate, the lipids were solubilized in Triton X-100. In mixtures of Triton with soy PC in the absence of PE, peroxidation dramatically increased the rate of lipid hydrolysis. In addition, the rate of hydrolysis of the unoxidizable lipid 1-palmitoyl-2-[1-¹⁴C]oleoyl PC incorporated into PC/PE liposomes was unaffected by peroxidation of the host lipid. These data are consistent with the notions that the increase in rate of hydrolysis of peroxidized PC substrates catalyzed by phospholipase A₂ is due largely to a preference for peroxidized phospholipid molecules as substrates and that peroxidation of host lipid does not significantly increase the rate of hydrolysis of nonoxidized lipids.     

Quantitation of vitamin K in human milk

A quantitative method was developed for the assay of vitamin K in human colostrum and milk. The procedure combines preparative and analytical chromatography on silica gel in a nitrogen atmosphere followed by reversed phase high performance liquid chromatography (HPLC). Two HPLC steps were used: gradient separation with ultraviolet (UV) detection followed by isocratic separation detected electrochemically. Due to co-migrating impurities, UV detection alone is insufficient for identification of vitamin K. Exogenous vitamin K was shown to equilibrate with endogenous vitamin K in the samples. A statistical method was incorporated to control for experimental variability. Vitamin K₁ was analyzed in 16 pooled milk samples from 7 donors and in individual samples from 15 donors at 1 month post-partrum. Vitamin K₁ was present at 2.94±1.94 and 3.15±2.87 ng/mL in pools and in individuals, respectively. Menaquinones, the bacterial form of the vitamin, were not detected. The significance of experimental variation to studies of vitamin K in individuals is discussed.     

Permeation and diffusion of environmental pollutants through flexible polymers

Permeation and diffusion characteristics of high density polyethylene (HDPE), elasticized polyolefin, and polychloroprene membranes have been determined for various organic pollutants at 22 and 50°C. A standard ASTM permeation cell method was used to measure the permeation rate while an immersion/weight gain method was used to obtain the diffusion coefficient. Thickness-dependent permeation rates were determined for HDPE membranes. An equimolar mixture of various permeants was also used to see the possibility of synergistic effects by combination of permeants. The experimental results are discussed in relation to the suitability of HDPE membranes in severe environments.     

Aggregation pheromone characterization and comparison inDrosophila ananassae andDrosophila bipectinata

(Z)-11-Octadecenyl acetate (Z11–18Ac) and (Z)-11-eicosenyl acetate (Z11–20Ac) were identified as the aggregation pheromones ofDrosophila ananassae, andZ11–20Ac was identified as the aggregation pheromone ofDrosophila bipectinata. Z11–18Ac andZ11–20Ac were not attractive alone; however, in combination with fermenting food odors, the acetates attracted flies of both sexes in a wind-tunnel olfactometer. The pheromones were present in the ejaculatory bulb of sexually mature male flies and transferred to the female during mating. MaleD. bipectinata released little if anyZ11–20Ac to the food; however, recently mated females releasedZ11–20Ac to the surrounding surfaces in just a few hours after mating.D. ananassae males, on the other hand, appeared to release moreZ11–18Ac andZ11–20Ac to the surroundings than mated females. AlthoughD. bipectinata males had noZ11–18Ac, flies were as attracted toZ11–18Ac as to an equal quantity ofZ11–20Ac.D. ananassae were attracted toZ11–18Ac but not toZ11–16Ac orZ11–20Ac. However,Z11–20Ac in combination withZ11–18Ac was significantly more attractive thanZ11–18Ac alone.     

Cuticular lipids of insects: V. Cuticular wax esters of secondary alcohols from the grasshoppersMelanoplus packardii andMelanoplus sanguinipes

Wax esters of secondary alcohols constitute 18–20% of the cuticular lipid extract ofMelanoplus packardii and 26–31% of the cuticular lipids ofMelanoplus sanguinipes. The total number of carbons in the wax esters range from 37–54 with 41 predominating in both species. The fatty acids ofM. packardii wax esters are 16∶0, 18∶0, 14∶0, 20∶0 and 12∶0 in decreasing quantity. The fatty acids ofM. sanguinipes wax esters are 18∶0, 20∶0, 16∶0 22∶0, 14∶0, 19∶0 and 17∶0 in decreasing quantity. The secondary alcohols from the wax esters ofM. packardii are C₂₅, C₂₃ and C₂₇ in decreasing quantity, and the secondary alcohols of theM. sanguinipes are C₂₃, C₂₅, C₂₁, C₂₇, C₂₄, C₂₂ and C₂₆ in decreasing quantity. Each secondary alcohol consists of two to four isomers with the hydroxyl group located near the center of the chain. Montana Agriculture Experiment Station, Journal Series No. 332.